Peptides and pharmaceutical, nutraceutical or veterinary compositions for hair loss prevention and/or treatment

ABSTRACT

Present invention discloses particular peptide sequences of formula (I); 
       (R 1 ) (m) -Xaa 1 IXaa 2 T(Q) (p) (R 2 ) (n)    (I) (SEQ ID NO: 1);
 
     wherein R 1 , m, p, n, Xaa 1 , Xaa 2 , and R 2  have particular meanings, or pharmaceutically or veterinary acceptable salts of these peptides that are effective in the prevention and/or treatment of mammal hair loss. 
     Invention also relates to particular topical pharmaceutical, nutraceutical or veterinary compositions with the peptides of formula (I).

This application claims the benefit of European Patent ApplicationEP16164645 filed on Apr. 11, 2016.

REFERENCE TO ELECTRONIC SEQUENCE LISTING

This application contains a sequence listing which has been submittedelectronically in .XML format and is hereby incorporated by reference inits entirety. Said .XML copy, created on Nov. 10, 2022, is named“108663_00315_ST26.xml” and is 30,880 bytes in size which is a copy ofthe sequence listing as filed in PCT/EP2017/057711. The sequence listingcontained in this .XML file is part of the specification and is herebyincorporated by reference herein in its entirety.

Present invention relates to the field of hair loss treatment of anycause. In particular, it relates to compounds and compositions,particularly topical formulations, comprising the peptides forameliorating alopecia and other hair diseases.

BACKGROUND ART

Many are the causes of hair loss, and several hair loss types are known,such as alopecia areata, androgenic alopecia, female-pattern alopecia,etc. In addition hair loss can be metabolic syndrome related, due tomedication (chemotherapy), a vitamin or mineral deficiency, or due toscalp fungal infections

Minoxidil (6-Piperidin-1-ylpyrimidine-2,4-diamine 3-oxide) is one of theactive principles mostly used and known in the treatment of alopecia, inparticular of androgenic alopecia. Common side effects include burningor irritation of the eye, itching, redness or irritation at the treatedarea, as well as unwanted hair growth elsewhere on the body. It wasdisclosed in U.S. Pat. No. 4,139,619 as an ingredient of a topicalcomposition for application to mammalian skin to increase the rate ofterminal hair growth. U.S. Pat. No. 4,596,812 discloses the particularuse of minoxidil in the treatment of alopecia.

Melatonin was also proposed for the treatment of androgenicfemale-pattern alopecia in U.S. Pat. No. 6,281,241, in particular due toits effect in reducing the telegenic rate and for occipital hair.

Besides, plant extracts are also proposed with the aim of applying morenatural compounds for the treatment of hair loss. As an example,publication of the patent application WO2007113851 proposed novelcompositions for hair loss prevention and/or hair growth promotion, inwhich several extract of Vernonia sp. were used as active agents againsthair diseases associated in the management of testosterone inducedandrogenic alopecia.

Other extracts, now of Curcuma sp and its active principle curcumin,seemed to avoid hair loss when administered together with resveratroland other ingredients as deduced from publication of the patentapplication WO2006087759. Table 1 in Example 1 of WO2006087759illustrated that some of the treated patients experienced hair growth inbald areas. Also mainly to curcumin present in Curcuma sp extracts, thedocument of Pumthong et al. “Curcuma aeruginosa, a novel botanicallyderived 5α-reductase inhibitor in the treatment of male-patternbaldness: a multicenter, randomized, double-blind, placebo-controlledstudy”, Journal of Dermatological Treatment. -2012; vol. no. 23, pp.:385-392, discloses the use of a Curcuma aeruginosa extract (hexaneextract) for treating androgenic alopecia. Although meaningful data wereachieved with combination of minoxidil and the Curcuma extract, theauthors postulated that due to its role as 5α-reductase inhibitor, asynergistic effect was observable also due to the direct stimulation ofhair growth produced by minoxidil.

Despite all these above-disclosed approaches for treating and/orpreventing hair loss, there is still a need of alternative active agentsand compositions comprising them for facing hair loss of any cause. Inparticular there is a need of active agents with proved efficacy, withminimized or null side effects and that can be manufactured in anenvironmentally and season independent way in case of being derived fromplants.

SUMMARY OF THE INVENTION

Inventors surprisingly found that the cell-free supernatants resultingfrom removal of entire cells in a plant dedifferentiated cell culturesuspension contain cocktails of compounds, acting synergisticallytogether with other extracellular products comprised in the supernatantfor the purpose of promoting hair growth and the proliferation of hairfollicle dermal papilla cells (HFDPC). Moreover, they proved thatpeptides comprised in isolated peptide fractions of said cell-freesupernatants, said peptides mainly involved in plant defence, growth anddevelopment were also effective as hair growth promoters and as hairfollicle dermal papilla cells proliferation and hair matrixkeratinocytes proliferation. Therefore, being both the peptides as wellas any fraction of a cell-free supernatant containing them useful in thetreatment of hair loss.

In a first aspect, the invention relates to peptide sequences of formula(I), or pharmaceutically, nutraceutical or veterinary acceptable saltsthereof,

(R¹)_((m))-Xaa₁IXaa₂T(Q)_((p))(R²)_((n))   (I) (SEQ ID NO: 1)

wherein:

R¹ is a —C(O)-(C₁-C₂₀)-alkyl radical, acylating N-terminal residue ofthe peptide sequence;

R² is a —NR₄R₅ radical, amidating C-terminal residue of the peptidesequence, being R₄ and R₅ selected from H, and (C₁-C₃)-alkyl;

m, n and p, are integers from 0 to 1; and

Xaa₁ and Xaa₂ are tyrosine residues with the side-chain hydroxyl groupoptionally replaced by a radical —OSO₃R³; being R³ selected from H and(C₁-C₃)-alkyl;

for use in the prevention and/or treatment of mammal hair loss in a hairloss causing disease and/or disorder.

This aspect could be also formulated as the use of a peptide sequence offormula (I) or a pharmaceutically, nutraceutical or veterinaryacceptable salt thereof, for the preparation of a medicament for theprophylaxis and/or treatment of a mammal hair loss in a mammal hair losscausing disease and/or disorder. It also relates to a method for theprophylaxis and/or treatment of hair loss in a mammal which comprisesadministering to mammals in need of such treatment an effective amountof a peptide sequence of formula (I) or a pharmaceutically,nutraceutical or veterinary acceptable salt thereof.

As illustrated in the examples below, the peptides of the inventionprovided a higher effect than minoxidil in terms of HFDPC proliferation.Besides, compositions comprising them, such as cell-free supernatants ofplants that previously supported the growth of a dedifferentiated plantcell suspension culture showed also equal or even higher effects thanminoxidil.

The peptides proposed for the use according to the invention arecompounds miming the phytosulfokine-β (PSK-β), isolated from conditionedmedia of plant cells in suspension (see Matsubayashi et al.,“Phytosulfokine, sulphated peptides that induce the proliferation ofsingle mesophyll cells of Asparagus officinalis L.”, Proc. Natl. Acad.Sci.- 1996, vol. no. 93, pp.: 7623-7627). To the best of inventor'sknowledge, this is the first time phytosulfokine-β (PSK-β) or aderivative of phytosulfokine-β (PSK-β) is proposed as a medicament, inparticular as hair loss preventing or treating agent.

A second aspect of the invention is a pharmaceutical, nutraceutical orveterinary composition comprising peptides of formula (I), or apharmaceutically, nutraceutical or veterinary acceptable salt thereof,together with pharmaceutically, nutraceutical or veterinary acceptableexcipients and/or carriers for use in the treatment hair loss in a hairloss causing disease and/or disorder.

Inventors have also developed particular effective compositions, inparticular topical compositions to be administered on scalp or thedesired bald area. These compositions comprise all those ingredientsallowing proliferation of HFDPC and provide to these proliferated cellsthe materials to allow hair growth. Hair growth is thus effected inscalp, but also in any place where the compositions are applied andwhere hair is usually present, such as in the lashes, the eyebrows andthe beard.

Thus, in a third aspect, the invention relates to pharmaceutical,nutraceutical or veterinary compositions comprising vitamins, aminoacids, pharmaceutically, nutraceutical or veterinary acceptable salts ofiron, magnesium, copper and zinc, together with pharmaceutically,nutraceutical or veterinary acceptable excipients and/or carriers, thecomposition further comprising

-   -   a peptide of formula (I), or a pharmaceutically, nutraceutical        or veterinary acceptable salt thereof, and/or    -   a cell-free supernatant of a dedifferentiated plant cell culture        suspension, or a fraction of said cell-free supernatant, wherein        said cell-free supernatant or said fraction comprises peptides        from 4 to 300 amino acids length, said peptides selected from        peptide plant growth factors, plant transcription factors,        epigenetic factors, and mixtures thereof, said peptide plant        growth factors comprising the peptide of formula (I), and said        cell-free supernatant or said fraction without having        cytoplasmic cell contents from the cell lysis and membranes        and/or cell walls.

Another aspect of the invention is a kit comprising:

(a) a peptide sequence of formula (I), or a pharmaceutically,nutraceutical or veterinary acceptable salt thereof; and

(b) a pharmaceutical, nutraceutical or veterinary composition comprisingvitamins, amino acids, and/or pharmaceutically, nutraceutical orveterinary acceptable salts thereof; pharmaceutically, nutraceutical orveterinary acceptable organic or inorganic salts of iron, magnesium,copper and zinc-, together with pharmaceutically, nutraceutical orveterinary acceptable excipients and/or carriers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 , related with Assay 1, is graph with the proliferation index (%)of hair follicle dermal papilla cells (HFDPC), when treated with severalcompositions. Ctrl BM means basal control (non-treated HFDPC), which isaccorded a 100% value of proliferation index: FGF means fibroblastgrowing factor, VEGF means vascular endothelial growing factor, C1 andC2 correspond to different concentrations of cell-free supernatants ofthe tested species, as indicated in Table 1, said supernatants (alsoknown as conditioned media) comprising peptides from 4 to 300 aminoacids length. Plant species names have been abbreviated also asindicated in Table 1. Peptide 4Aa and 5Aa are peptides of SEQ ID NO: 6and 7, respectively. In Y-axis the proliferation index in percentage(%).

FIG. 2 , related with Assay 1, is a graph with the proliferation index(%) of hair follicle dermal papilla cells (HFDPC), when treated with thepeptide 4Aa carrying various end-terminal modifications. “Basal Ctrl”means basal control (non-treated HFDPC), which is accorded a 100% valueof proliferation index. C1, C2 and C3 represent different concentrationsof peptides, corresponding to 5.86 μg/ml, 2.93 μg/ml and 1.46 μg/ml,respectively. The first column corresponds to the 4Aa peptide whereinboth ends are free (SEQ ID NO:12); the second column corresponds to the4Aa peptide wherein the N-terminal end is acetylated the C-terminal endis free (SEQ ID NO:16); the third column corresponds to the 4Aa peptidewherein the N-terminal end is free and the C-terminal end is amidated(SEQ ID NO:18); and the forth column corresponds to the 4Aa peptidewherein the N-terminal end is acetylated and the C-terminal end isamidated (SEQ ID NO: 6).

FIG. 3 , related with Assay 1, is a graph with the proliferation index(%) of hair follicle dermal papilla cells (HFDPC), when treated with thepeptide 5Aa carrying various end-terminal modifications. “Basal Ctrl”means basal control (non-treated HFDPC), which is accorded a 100% valueof proliferation index. C1, C2 and C3 represent different concentrationsof peptides, corresponding to 5.86 μg/ml, 2.93 μg/ml and 1.46 μg/ml,respectively. The first column corresponds to the 5Aa peptide whereinboth ends are free (SEQ ID NO:13); the second column corresponds to the5Aa peptide wherein the N-terminal end is acetylated the C-terminal endis free (SEQ ID NO:17); the third column corresponds to the 5Aa peptidewherein the N-terminal end is acetylated and the C-terminal end isamidated (SEQ ID NO: 7).

FIG. 4 , related with Assay 1, is an HPLC chromatogram of a DaucusCarota cell-free supernatant and a peptide of SEQ ID NO: 6. Y-axis,intensity of peaks, X-axis, retention time (T) in minutes (min).

FIG. 5 , related with Assay 1, is a graph showing the expression levelsof miRNA-22 (FIG. 5 (B)) and miRNA-31 (FIG. 5 (A) and (C)) in secretomasfrom HFDPC treated with either the peptide of SEQ ID NO: 6 or thecell-free supernatants of Curcuma longa.

FIG. 6 , related with Assay 2, is a graph with the proliferation index(%) of hair dermal fibroblasts (HDF), when treated with the peptide 4Aacarrying various end-terminal modifications. “Basal Ctrl” means basalcontrol (non-treated HDF), which is accorded a 100% value ofproliferation index. C1, C2 and C3 represent different concentrations ofpeptides, corresponding to 25 μg/ml, 12.5 μg/ml and 1.25 μg/ml,respectively. The first column corresponds to the 4Aa peptide whereinboth ends are free (SEQ ID NO:12); the second column corresponds to the4Aa peptide wherein the N-terminal end is acetylated the C-terminal endis free (SEQ ID NO:16); the third column corresponds to the 4Aa peptidewherein the N-terminal end is free and the C-terminal end is amidated(SEQ ID NO:18); and the forth column corresponds to the 4Aa peptidewherein the N-terminal end is acetylated and the C-terminal end isamidated (SEQ ID NO: 6).

FIG. 7 , related with Assay 2, is a graph with the proliferation index(%) of hair dermal fibroblasts (HDF), when treated with the peptide 5Aacarrying various end-terminal modifications. “Basal Ctrl” means basalcontrol (non-treated HDF), which is accorded a 100% value ofproliferation index. C1, C2 and C3 represent different concentrations ofpeptides, corresponding to 1.25 μg/ml, 0.63 μg/ml and 0.25 μg/ml,respectively. The first column corresponds to the 5Aa peptide whereinboth ends are free (SEQ ID NO:13); the second column corresponds to the5Aa peptide wherein the N-terminal end is acetylated the C-terminal endis free (SEQ ID NO:17); the third column corresponds to the 5Aa peptidewherein the N-terminal end is free and the C-terminal end is amidated(SEQ ID NO:19); and the forth column corresponds to the 5Aa peptidewherein the N-terminal end is acetylated and the C-terminal end isamidated (SEQ ID NO: 7).

FIG. 8 , and FIG. 9 , related with Assay 3, depict scalp pictures ofoccipital (A) and frontal (B) parts, and capillary densitymicrophotographies (C) made with Trichoscan® of volunteers receivingpeptide of SEQ ID NO: 6 (FIG. 8 ), or a cell free supernatant of Curcumalonga comprising peptides of formula (I) (FIG. 9 ).

FIG. 10 , related with Assay 4, is a graph with the proliferation index(%) of hair follicle dermal papilla cells (HFDPC), when treated with afortifying composition at different concentrations. “Basal Ctrl” meansbasal control (non-treated HDFPC), which is accorded a 100% value ofproliferation index.

FIG. 11 , related with Assay 4, is a graph with the proliferation index(%) of hair dermal fibroblasts (HDF), when treated with a fortifyingcomposition at different concentrations. “Basal Ctrl” means basalcontrol (non-treated HDF), which is accorded a 100% value ofproliferation index.

FIG. 12 , related with Assay 4, is a graph with the proliferation index(%) of human immortalized keratinocytes (HaCat), when treated with afortifying composition at different concentrations. “Basal Ctrl” meansbasal control (non-treated HDFPC), which is accorded a 100% value ofproliferation index.

FIG. 13 , related with Assay 4, is a graph with the proliferation index(%) of hair follicle dermal papilla cells (HFDPC), when treated with afortifying composition different of that of FIGS. 10 and 12 , atdifferent concentrations. “Basal Ctrl” means basal control (non-treatedHDFPC), which is accorded a 100% value of proliferation index.

FIG. 14 , related with Assay 4, is a graph with the proliferation index(%) of hair dermal fibroblasts (HDF), when treated with the fortifyingcomposition as in FIG. 13 at different concentrations. “Basal Ctrl”means basal control (non-treated HDF), which is accorded a 100% value ofproliferation index.

FIG. 15 , related with Assay 4, is a graph with the proliferation index(%) of human immortalized keratinocytes (HaCat), when treated with thefortifying composition as in FIG. 13 at different concentrations. “BasalCtrl” means basal control (non-treated HDFPC), which is accorded a 100%value of proliferation index.

FIG. 16 , related with Assay 5, is a graph with the proliferation index(%) of hair follicle dermal papilla cells (HFDPC), when treated with a4Aa peptide of formula (I), with a fortifying composition, or with acombination of both. “Basal Ctrl” means basal control (non-treatedHDFPC), which is accorded a 100% value of proliferation index.

FIG. 17 , related with Assay 6 (reference Example), is a graph depictingthe wound healing potential of hair dermal fibroblasts (HDF) treatedwith the peptide 4Aa carrying various sulfation modifications. “BasalCtrl” means basal control and consists of HDF treated with 0,1% FBS. Twopositive controls are represented, either treated with 10% FBS or TGF-β1(15 ng/ml). The peptide 4AaS1 consists on a modified version of the 4Aapeptide, which carries a —OSO₃H radical in the first tyrosine of thepeptide (SEQ ID NO: 2); and the peptide 4AaS2 consists on a modifiedversion of the 4Aa peptide, which carries a —OSO₃H radical in the secondtyrosine of the peptide (SEQ ID NO: 3).

DETAILED DESCRIPTION OF THE INVENTION

All terms as used herein in this application, unless otherwise stated,shall be understood in their ordinary meaning as known in the art. Othermore specific definitions for certain terms as used in the presentapplication are as set forth below and are intended to apply uniformlythrough-out the specification and claims unless an otherwise expresslyset out definition provides a broader definition.

For “mammal hair loss in a hair loss causing disease” it is to beunderstood that hair is lost or changed in a frequency more than thatassociated with health or standard hair renewal, or that hair isweakened, or it does not grown again after having fallen, being said“hair loss” caused by any disorder or disease, which symptom orconsequence is that the number of hair in a particular area is decreasedor the number of hair in a particular area is a weak hair. The “hairloss” may also be due to any other circumstances, such as the medicationside effects. In particular the causes, diseases or disorders causinghair loss are to be understood as encompassing: alopecia (i.e.androgenic alopecia, androgenic female-pattern alopecia, alopeciaareata, seasonal alopecia, diffuse alopecia, metabolic syndromerelated), medication (i.e. chemotherapy), hypotrichosis, vitamin ormineral deficiency, trichotillomania, hypothyroidism, tightly pulledhair, scalp fungal infections, hair loss due to technical processes (dueto stressing hair treatments, such as staining), hair fragility andmenopause, and combinations thereof.

As used herein, the term “pharmaceutically, nutraceutical or veterinaryacceptable salt” refers to those salts which are, within the scope ofsound medical judgment, suitable for use in contact with the tissues ofhumans and lower animals without undue toxicity, irritation, allergicresponse and the like, and are commensurate with a reasonablebenefit/risk ratio. Pharmaceutically acceptable salts are well known inthe art. Examples of pharmaceutically acceptable, nontoxic acid additionsalts are salts of an amino group formed with inorganic acids such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid andperchloric acid or with organic acids such as acetic acid,trifluoroacetic acid, oxalic acid, maleic acid, tartaric acid, citricacid, succinic acid or malonic acid or by using other methods used inthe art such as ion exchange. Other pharmaceutically acceptable saltsinclude adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate,citrate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate,gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, and ammonium. Representative alkali or alkalineearth metal salts include sodium, lithium, potassium, calcium,magnesium, and the like. Further pharmaceutically acceptable saltsinclude, when appropriate, nontoxic ammonium, quaternary ammonium, andamine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and arylsulfonate.

“Nutraceutical compositions” are edible compositions including dietarysupplements, oral rehydration solutions (ORS), food additives, babycereals and infant formulas. Dietary supplements intend to supplynutrients, (vitamins, minerals, fatty acids or amino acids) that aremissing or not consumed in sufficient quantity in a person's diet(infants, pregnant women, elderly people, etc).

The term “pharmaceutically, nutraceutical or veterinary effectiveamount” as used herein, means an amount of an active agent high enoughto deliver the desired benefit, but low enough to avoid serious sideeffects within the scope of medical or veterinary judgment.

The term (C₁-C₃)alkyl refers to a saturated straight or branched alkylchain having from 1 to 3 carbon atoms. Illustrative non-limitativeexamples are: methyl, ethyl, propyl, and isopropyl.

The term —C(O)-(C₁-C₂₀)-alkyl radical, refers to a saturated straight orbranched alkanoyl acid chain having from 2 to 21 carbon atoms.Illustrative non-limitative examples are: acetyl, propanoyl,isopropanoyl, butanoyl, isobutanoyl, sec-butanoyl, tert-butanoyl,n-pentanoyl, neo-pentanoyl, n-hexanoyl, n-myristoyl (or n-tetradecanoil)and n-palmitoyl (or n-hexadecanoyl). Particular —C(O)-(C₁-C₂₀)-alkylradicals in the present invention are acetyl, n-myristoyl (orn-tetradecanoil) and n-palmitoyl (or n-hexadecanoyl).

The term “amino acid” constituting the peptide sequences refers to amolecule containing both an amino group and a carboxyl group. In certainembodiments, an amino acid is an alpha amino acid. Suitable amino acidsinclude, without limitation, natural alpha-amino acids such as L-isomersof the 20 common naturally occurring alpha-amino acids. Amino acids usedin the construction of peptides of the present invention may be preparedby organic synthesis, or obtained by other routes, such as, for example,degradation of or isolation from a natural source.

When in the present invention it is indicated that the tyrosine residuesare ones in which their side-chain hydroxyl group has been replaced by aradical —OSO₃R³; being R³ selected from H and (C₁-C₃)-alkyl, it is to beunderstood as encompassing those sulphated tyrosines (—OSO3H) andpharmaceutically acceptable salts thereof (due to ionic equilibrium withcounter-ions). Tyrosine sulphation is a naturally occurringposttranslational modification where a sulphate group is added to atyrosine residue of a protein molecule. Sulphation is catalyzed bytyrosylprotein sulfotransferase (TPST) in the Golgi apparatus. Thereaction catalyzed by TPST is a transfer of sulphate from the universalsulphate donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to theside-chain hydroxyl group of a tyrosine residue. In synthetic peptideswith sulphated tyrosines, enzymatic reactions may be employed, as wellas chemical sulphation reactions widely known by the skilled man.

The expression “cell-free supernatant that previously supported thegrowth of a dedifferentiated plant cell suspension culture” refers onlyto the liquid that contained the dedifferentiated plant cells duringgrowing or cultivation in suspension in a culture medium and for anypurpose (production of secondary metabolites, biomass production, etc.).The cell-free supernatant does not contain the cytoplasm contentsreleased from the lysis of the plant cells, as well as any part of theplant cell resulting from disruption of the same (membrane fragments,cell-wall fragments, etc.). The cell-free supernatant comprises amongthe ingredients of the initial culture medium that have been notconsumed, also those compounds secreted by the plant cells to theextracellular media. Among these compounds there are the peptide plantgrowth factors, transcription factors and epigenetic factors. Thecell-free supernatant is also called conditioned nutrient media orconditioned media, or conditioned cell-medium (used herewithinterchangeably). When it is said that the cell-free supernatants are“without having cytoplasmic cell contents from the cell lysis andwithout having membranes and/or cell walls” is to be understood thattraces of some membranes and/or cell walls, and cytoplasmic components(nucleic components, organelles, etc.) can be present due to thespontaneous disruption of isolated cells occurring occasionally duringthe culturing process.

In a particular embodiment, optionally in combination with anyembodiments below, the peptides of formula (I) for use in the preventionand/or treatment of hair loss in a mammal hair-loss causing disease arethose, wherein R¹ is a —C(O)—(C₁-C₂₀)-alkyl radical selected from—C(O)—CH₃ radical, n-myristoyl, and n-palmitoyl.

In another particular embodiment, optionally in combination with anyembodiments above or below, the peptides of formula (I) for use in theprevention and/or treatment of hair loss in a mammal hair-loss causingdisease are those, wherein R³ of —OSO₃R³ is hydrogen (H), encompassingalso salts of alkaline metals of the acid form.

In a further particular embodiment the peptides of formula (I) for usein the prevention and/or treatment of hair loss in a mammal hair-losscausing disease are those wherein m and n are 1 and p is from 0 to 1.More in particular m and n are 1 and p is from 0 to 1, and R¹ is a—C(O)—CH₃ radical, R² is a —NH₂ radical, and R₃ is hydrogen (H).

Examples of these peptides are CH₃—C(O)-Xaa₁IYT-NH₂ (SEQ ID NO: 2),wherein Xaa₁ is a tyrosine residue with the side-chain hydroxyl groupreplaced by a radical —OSO₃H;

CH₃—C(O)-YIXaa₂T-NH₂ (SEQ ID NO: 3), wherein Xaa₂ is a tyrosine residuewith the side-chain hydroxyl group replaced by a radical —OSO₃H;

CH₃—C(O)-Xaa₁IYTQ-NH₂ (SEQ ID NO: 4) wherein Xaa₁ is a tyrosine residuewith the side-chain hydroxyl group replaced by a radical —OSO₃H;

CH₃—C(O)-YIXaa₂TQ-NH₂ (SEQ ID NO: 5), wherein Xaa₂ is a tyrosine residuewith the side-chain hydroxyl group replaced by a radical —OSO₃H;

CH₃—C(O)-YIYT-NH₂ (SEQ ID NO: 6); and

CH₃—C(O)-YIYTQ-NH₂ (SEQ ID NO: 7);

In a further particular embodiment the peptides of formula (I) for usein the prevention and/or treatment of hair loss in a mammal hair-losscausing disease are those wherein m is 1, n is 0, and p is from 0 to 1.More in particular wherein m is 1, n is 0, and p is from 0 to 1 and R¹is a —C(O)—CH₃ radical, and R³ is H. Examples of these peptides offormula (I) are CH₃—C(O)-YIYT (SEQ ID NO: 16); and CH₃—C(O)-YIYTQ (SEQID NO: 17).

In a further particular embodiment the peptides of formula (I) for usein the prevention and/or treatment of hair loss in a mammal hair-losscausing disease are those wherein m is 0, n is 1, and p is from 0 to 1.More in particular, wherein m is 0, n is 1, and p is from 0 to 1, R² isa —NH₂ radical, and R³ is H. Examples of these peptides of formula (I)are YIYT-NH₂ (SEQ ID NO: 18); and YIYTQ-NH₂ (SEQ ID NO: 19).

Also in a further particular embodiment the peptides of formula (I) foruse in the prevention and/or treatment of hair loss in a mammalhair-loss causing disease are those wherein m and n are both 0, and p isfrom 0 to 1. Examples of these peptides of formula (I) are Xaa₁IYT (SEQID NO: 8), wherein Xaa₁ is a tyrosine residue with the side-chainhydroxyl group replaced by a radical—OSO₃H; YIXaa₂T (SEQ ID NO: 9),wherein Xaa₂ is a tyrosine residue with the side-chain hydroxyl groupreplaced by a radical —OSO₃H; Xaa₁IYTQ (SEQ ID NO: 10) wherein Xaa₁ is atyrosine residue with the side-chain hydroxyl group replaced by aradical —OSO3H; YIXaa2TQ (SEQ ID NO: 11), wherein Xaa₂ is a tyrosineresidue with the side-chain hydroxyl group replaced by a radical —OSO₃H;YIYT (SEQ ID NO: 12); YIYTQ (SEQ ID NO: 13); Xaa₁IXaa₂T (SEQ ID NO: 14),wherein Xaa₁ and Xaa₂ are both a tyrosine residue with the side-chainhydroxyl group replaced by a radical —OSO₃H; and Xaa₁IXaa₂TQ (SEQ ID NO:15), wherein Xaa₁ and Xaa₂ are both a tyrosine residue with theside-chain hydroxyl group replaced by a radical —OSO₃H

In other certain embodiments, optionally in combination with anyembodiment above or below, the peptides for use according to theinvention are selected from the group consisting of:

CH₃—C(O)-Xaa₁IYT-NH₂ (SEQ ID NO: 2), wherein Xaa₁ is a tyrosine residuewith the side-chain hydroxyl group replaced by a radical —OSO₃H;

CH₃—C(O)-YIXaa₂T-NH₂ (SEQ ID NO: 3), wherein Xaa₂ is a tyrosine residuewith the side-chain hydroxyl group replaced by a radical —OSO₃H;

CH₃—C(O)-Xaa₁IYTQ-NH₂ (SEQ ID NO: 4) wherein Xaa₁ is a tyrosine residuewith the side-chain hydroxyl group replaced by a radical —OSO₃H;

CH₃—C(O)-YIXaa₂TQ-NH2 (SEQ ID NO: 5), wherein Xaa₂ is a tyrosine residuewith the side-chain hydroxyl group replaced by a radical —OSO₃H;

CH₃—C(O)-YIYT-NH₂ (SEQ ID NO: 6);

CH₃—C(O)-YIYTQ-NH₂ (SEQ ID NO: 7);

Xaa₁ IYT (SEQ ID NO: 8), wherein Xaa₁ is a tyrosine residue with theside-chain hydroxyl group replaced by a radical —OSO₃H;

YIXaa₂T (SEQ ID NO: 9), wherein Xaa₂ is a tyrosine residue with theside-chain hydroxyl group replaced by a radical —OSO₃H;

Xaa₁IYTQ (SEQ ID NO: 10) wherein Xaa₁ is a tyrosine residue with theside-chain hydroxyl group replaced by a radical —OSO₃H;

YIXaa₂TQ (SEQ ID NO: 11), wherein Xaa₂ is a tyrosine residue with theside-chain hydroxyl group replaced by a radical —OSO₃H;

(SEQ ID NO: 12) YIYT; (SEQ ID NO: 13) YIYTQ;

Xaa₁IXaa₂T (SEQ ID NO: 14), wherein Xaa₁ and Xaa₂ are both a tyrosineresidue with the side-chain hydroxyl group replaced by a radical —OSO₃H;

Xaa1IXaa₂TQ (SEQ ID NO: 15), wherein Xaa₁ and Xaa₂ are both a tyrosineresidue with the side-chain hydroxyl group replaced by a radical —OSO₃H;

(SEQ ID NO: 16) CH_(3—)C(O)- YIYT; (SEQ ID NO: 17) CH_(3—)C(O)- YIYTQ;(SEQ ID NO: 18) YIYT-NH₂; (SEQ ID NO: 19) YIYTQ-NH₂;

and mixtures thereof.

In a more particular embodiment, the peptides are selected from thegroup consisting of:

(SEQ ID NO: 6) CH₃—C(O)- YIYT-NH₂; (SEQ ID NO: 7)CH_(3—)C(O)- YIYTQ-NH₂;

Xaa₁IYT (SEQ ID NO: 8), wherein Xaa₁ is a tyrosine residue with theside-chain hydroxyl group replaced by a radical —OSO₃H;

YIXaa₂T (SEQ ID NO: 9), wherein Xaa₂ is a tyrosine residue with theside-chain hydroxyl group replaced by a radical —OSO₃H;

(SEQ ID NO: 12) YIYT; (SEQ ID NO: 13) YIYTQ;

Xaa₁IXaa₂T (SEQ ID NO: 14), wherein Xaa₁ and Xaa₂ are both a tyrosineresidue with the side-chain hydroxyl group replaced by a radical —OSO₃H;

Xaa1IXaa₂TQ (SEQ ID NO: 15), wherein Xaa₁ and Xaa₂ are both a tyrosineresidue with the side-chain hydroxyl group replaced by a radical —OSO₃H;

and mixtures thereof.

All these peptides of formula (I) can be formulated in pharmaceutical,nutraceutical or veterinary compositions, wherein they are one of theactives against hair loss. The invention relates, as above exposed, alsoto those pharmaceutical, nutraceutical or veterinary compositionscomprising peptides of formula (I), or a pharmaceutically, nutraceuticalor veterinary acceptable salt thereof, together with pharmaceutically orveterinary acceptable excipients and/or carriers for use in theprevention and/or treatment of a hair loss in a hair loss causingdisease.

When formulated in pharmaceutical, nutraceutical or veterinarycompositions, the peptides of formula (I) may be, in a particularembodiment, conveniently protected against proteases, and/or preparedfor penetrating the cells. Agents that facilitate the delivery of thepeptide of the invention across a cell membrane without negativelyaffecting the ability of the peptide to promote cell proliferation andhair growth include penetrating peptides fused or bound thereto;liposomes; and nanoparticles (1-300 nm).

In a particular embodiment of this second aspect, the compositions forthe purposed use are topical compositions that are applied onto scalpand/or to bald regions. Particular topical compositions are selectedfrom the group consisting of solutions, creams, lotions, unguents,emulsions, aerosols and non-aerosol sprays, gels, ointments andsuspensions, all of them rinse-off and not rinsed compositions. Hairgrowth is thus effected in any place where the compositions are appliedand where hair is usually present, such as in the lashes, the eyebrows,the scalp and the beard.

In another particular embodiment of the second aspect thepharmaceutical, nutraceutical or veterinary compositions for thepurposed use further comprise vitamins, amino acids, pharmaceutically orveterinary acceptable organic or inorganic salts of iron, magnesium,copper and zinc.

Indeed, these compositions further comprising the vitamins, metal saltsand amino acids, provide to the treated area or to the area where theyare applied the mixture of cell nutrients (as a reinforcing composition)that allow papilla cells and papilla matrix (keratinocytes) forming orproducing new hair.

In a more particular embodiment the pharmaceutical, nutraceutical orveterinary compositions for the purposed use comprise the vitaminsselected from the group consisting of vitamin C, vitamin B1, vitamin B3,vitamin B5, vitamin B6, vitamin B2, biotin (which is the water solublevit B8), vitamin B9, myo-inositol, and mixtures thereof.

In another also more particular embodiment, the pharmaceutical,nutraceutical or veterinary compositions for the purposed use compriseamino acids, or salts thereof, selected from the group consisting ofglutamic acid, phenylalanine, glycine, methionine, proline, cysteine andmixtures thereof. In another particular embodiment protein hydrolysates,more in particular casein hydrolysates, are comprised therein.

In another particular embodiment, the pharmaceutical, nutraceutical orveterinary composition for use as above indicated, is that in which theweight ratio of iron to copper is from 25:1 to 900:1, of zinc to copperis from 30:1 to 400:1, and of magnesium to copper is from 570:1 to5800:1. Weight ratio between elements relates to the weight of theelement in the formulation. In another more particular embodiment, incombination with the embodiments above or below, the weight ratio ofiron to copper is from 700:1 to 900:1, of zinc to copper is from 300:1to 400:1, and of magnesium to copper is from 2300:1 to 5800:1.Particular weight ratios of all the elements as Fe/Cu/Zn/Mg are:868/1/307/5730; and 25/1/307/2856.

Yet in another also more particular embodiment, the pharmaceutical,nutraceutical or veterinary compositions for the purposed use compriseas the acceptable salts of iron, magnesium, copper and zinc theinorganic salts selected from the group consisting of iron sulphate,copper sulphate, zinc sulphate, magnesium sulphate, and mixturesthereof.

In another particular embodiment, the pharmaceutical, nutraceutical orveterinary composition for use as above indicated, comprises a peptideof formula (I), or a pharmaceutically or veterinary acceptable saltthereof, vitamin C, vitamin B1, vitamin B2, vitamin B3, vitamin B5,vitamin B6, vitamin B8, vitamin B9, glycine, methionine, phenylalanine,proline, cysteine, glutamic acid, zinc sulphate, copper sulphate,magnesium sulphate and iron sulphate together with pharmaceutically,nutraceutical or veterinary acceptable excipients and/or carriers.

In a particular embodiment of the pharmaceutical, nutraceutical orveterinary composition for use as above disclosed, and also optionallyin combination with any embodiments above or below, the peptide offormula (I) is provided in the composition as an ingredient comprisedin, or of a cell-free supernatant that previously supported the growthof a dedifferentiated plant cell suspension culture, or a fraction ofsaid cell-free supernatant, wherein said cell-free supernatant or saidfraction comprises peptides from 4 to 300 amino acids length, saidpeptides selected from peptide plant growth factors, plant transcriptionfactors, epigenetic factors and mixtures thereof, and said cell-freesupernatant or said fraction without having cytoplasmic cell contentsfrom the cell lysis and membranes and/or cell walls.

Many of these cell-free supernatants or fractions thereof arecommercially available.

They can also be generically obtained by a method comprising:

(a) growing dedifferentiated plant cells from a friable callus in aliquid nutrient culture medium from 5 to 15 days, to obtain aconditioned media supporting the growth of the dedifferentiated plantcells;

(b) removing entire plant cells from the conditioned media withoutlysing the cells, to obtain a cell-free supernatant comprising peptidesfrom 4 to 300 amino acids length; and

(c) optionally carrying out a protein separation process by means of aseparation technique selected from the group consisting ofchromatography, filtration, ultrafiltration, protein precipitation, andcombinations thereof, to obtain a fraction of the cell-free supernatant.

In another particular embodiment, the pharmaceutical, nutraceutical orveterinary composition for use as above proposed, comprises the peptideof formula (I) provided in the composition as an ingredient of acell-free supernatant of a dedifferentiated plant cell culturesuspension from a plant selected from the group consisting of Daucuscarota, Centella asiatica, Sarcocapnos crassifolia, Curcuma longa, Vitisvinifera, Lithops pseudotruncatella, Morinda citrifolia, and Oleaeuropaea.

Indeed, these cell-free supernatants or fractions thereof comprisingpeptides from 4 to 300 amino acids length may be the natural source ofthe peptides of formula (I), which in the examples are demonstrated ashighly active.

In another particular embodiment of the first and second aspects, thepeptide sequence of formula (I) or a pharmaceutically, nutraceutical orveterinary acceptable salt thereof, or the pharmaceutical, nutraceuticalor veterinary composition comprising it, are for use in the preventionand/or treatment of mammal hair loss (in particular human hair loss) ina mammal hair loss causing disease or disorder selected from alopecia,medication (in particular chemotherapy), hypotrichosis, vitamin ormineral deficiency, trichotillomania, hypothyroidism, tightly pulledhair, scalp fungal infections, hair loss due to technical processes,hair fragility, menopause, and combinations thereof.

In another particular embodiment, the peptide sequence of formula (I) ora pharmaceutically, nutraceutical or veterinary acceptable salt thereof,or the pharmaceutical, nutraceutical or veterinary compositioncomprising it, are for use in the prevention and/or treatment of mammalhair loss of scalp, beard, lashes and eyebrows. This means that they arefor use also for promoting hair growth in beard, for elongating lashesand for increasing density of hair in the eyebrow area.

In particular, the peptides of formula (I), salts thereof, or anycomposition comprising them are for use in the prevention and/ortreatment of alopecia selected from androgenic alopecia, androgenicfemale-pattern alopecia, alopecia areata, seasonal alopecia, diffusealopecia, metabolic syndrome related alopecia.

According to the third aspect, the invention relates to pharmaceutical,nutraceutical or veterinary compositions comprising vitamins, aminoacids, pharmaceutically, nutraceutical or veterinary acceptable organicor inorganic salts of iron, magnesium, copper and zinc, together withpharmaceutically, nutraceutical or veterinary acceptable excipientsand/or carriers, the compositions further comprising:

-   -   a peptide of formula (I), or a pharmaceutically, nutraceutical        or veterinary acceptable salt thereof, and/or    -   a cell-free supernatant of a dedifferentiated plant cell culture        suspension, or a fraction of said cell-free supernatant, wherein        said cell-free supernatant or said fraction comprises peptides        from 4 to 300 amino acids length, said peptides selected from        peptide plant growth factors, plant transcription factors,        epigenetic factors, and mixtures thereof, said peptide plant        growth factors comprising the peptide of formula (I), and said        cell-free supernatant or said fraction without having        cytoplasmic cell contents from the cell lysis and membranes        and/or cell walls.

The invention relates, in particular, also to new topical pharmaceuticalor veterinary compositions comprising vitamins, amino acids,pharmaceutically acceptable organic or inorganic salts of iron,magnesium, copper and zinc, together with pharmaceutically or veterinaryacceptable excipients and/or carriers, the composition furthercomprising:

-   -   a peptide of formula (I), or a pharmaceutically or veterinary        acceptable salt thereof, and/or    -   a cell-free supernatant of a dedifferentiated plant cell culture        suspension, or a fraction of said cell-free supernatant, wherein        said cell-free supernatant or said fraction comprises peptides        from 4 to 300 amino acids length, said peptides selected from        peptide plant growth factors, plant transcription factors, plant        epigenetic factors and mixtures thereof, said peptide plant        growth factors comprising the peptide of formula (I), and said        cell-free supernatant or said fraction without having        cytoplasmic cell contents from the cell lysis and membranes        and/or cell walls.

In a particular embodiment of these pharmaceutical, nutraceutical orveterinary compositions, and so also of the topical pharmaceutical orveterinary compositions, the cell-free supernatant of a dedifferentiatedplant cell culture suspension, or a fraction of said cell-freesupernatant are from a plant selected from the group consisting ofDaucus carota, Centella asiatica, Sarcocapnos crassifolia, Curcumalonga, Vitis vinifera, Lithops pseudotruncatella, Morinda citrifolia,and Olea europaea.

In another particular embodiment of the pharmaceutical, nutraceutical orveterinary compositions, and so also of the topical pharmaceutical orveterinary compositions according to the invention, the peptide offormula (I) is particularly selected from:

CH₃—C(O)-YIYT-NH₂ (SEQ ID NO: 6);

CH₃—C(O)-YIYTQ-NH₂ (SEQ ID NO: 7);

Xaa₁ IYT (SEQ ID NO: 8), wherein Xaa₁ is a tyrosine residue with theside-chain hydroxyl group replaced by a radical —OSO₃H;

YIXaa₂T (SEQ ID NO: 9), wherein Xaa₂ is a tyrosine residue with theside-chain hydroxyl group replaced by a radical —OSO₃H;

YIYT (SEQ ID NO: 12);

YIYTQ (SEQ ID NO: 13);

Xaa₁IXaa₂T (SEQ ID NO: 14), wherein Xaa₁ and Xaa₂ are both a tyrosineresidue with the side-chain hydroxyl group replaced by a radical —OSO₃H;

Xaa₁IXaa₂TQ (SEQ ID NO: 15), wherein Xaa₁ and Xaa₂ are both a tyrosineresidue with the side-chain hydroxyl group replaced by a radical —OSO₃H;

CH₃—C(O)-YIYT (SEQ ID NO: 16);

CH₃—C(O)-YIYTQ (SEQ ID NO: 17);

YIYT-NH₂ (SEQ ID NO: 18);

YIYTQ-NH₂ (SEQ ID NO: 19);

and mixtures thereof.

In a more particular embodiment of the pharmaceutical, nutraceutical orveterinary compositions, and so also of the topical pharmaceutical orveterinary compositions according to the invention, it comprises apeptide of formula (I), or a pharmaceutically, nutraceutical orveterinary acceptable salt thereof; vitamins, amino acids, andpharmaceutically, nutraceutical or veterinary acceptable salts of iron,magnesium, copper and zinc, wherein the weight ratio of iron to copperis from 25:1 to 900:1, of zinc to copper is from 30:1 to 400:1, and ofmagnesium to copper is from 570:1 to 5800:1.

In also another particular embodiment of this aspect of the invention,the pharmaceutical, nutraceutical or veterinary composition comprises apeptide of formula (I), or a pharmaceutically or veterinary acceptablesalt thereof; vitamin C, vitamin B1, vitamin B2, vitamin B3, vitamin B5,vitamin B6, vitamin B8, vitamin B9, glycine, methionine, phenylalanine,proline, cysteine, glutamic acid, zinc sulphate, copper sulphate,magnesium sulphate and iron sulphate together with pharmaceutically,nutraceutical or veterinary acceptable excipients and/or carriers. Inyet a more particular embodiment, the peptide of formula (I) is selectedfrom CH₃—C(O)-YIYT-NH₂ (SEQ ID NO: 6); CH₃—C(O)-YIYTQ-NH₂ (SEQ ID NO:7);

Xaa₁IYT (SEQ ID NO: 8), wherein Xaa₁ is a tyrosine residue with theside-chain hydroxyl group replaced by a radical —OSO₃H;

YIXaa₂T (SEQ ID NO: 9), wherein Xaa₂ is a tyrosine residue with theside-chain hydroxyl group replaced by a radical —OSO₃H;

YIYT (SEQ ID NO: 12);

YIYTQ (SEQ ID NO: 13);

Xaa₁IXaa₂T (SEQ ID NO: 14), wherein Xaa₁ and Xaa₂ are both a tyrosineresidue with the side-chain hydroxyl group replaced by a radical —OSO₃H;

Xaa₁IXaa₂TQ (SEQ ID NO: 15), wherein Xaa₁ and Xaa₂ are both a tyrosineresidue with the side-chain hydroxyl group replaced by a radical —OSO₃H;

CH₃—C(O)-YIYT (SEQ ID NO: 16);

CH₃—C(O)-YIYTQ (SEQ ID NO: 17);

YIYT-NH₂ (SEQ ID NO: 18);

YIYTQ-NH₂ (SEQ ID NO: 19);

and mixtures thereof. More in particular is SEQ ID NO: 6

The combination of the peptides of formula (I) and the compositions(solutions) comprising salts of iron, copper, magnesium and zinc,together with vitamins and amino acids of the invention, has asynergistic effect in terms of proliferation of human dermal fibroblasts(HDF). This makes the combination useful in the treatment of hair lossbut also as tissue regeneration agent.

As indicated, one aspect of the invention is a kit comprising:

(a) a peptide sequence of formula (I), or a pharmaceutically,nutraceutical or veterinary acceptable salt thereof; and

(b) a pharmaceutical, nutraceutical or veterinary composition comprisingvitamins, amino acids, and/or pharmaceutically, nutraceutical orveterinary acceptable salts thereof; pharmaceutically, nutraceutical orveterinary acceptable organic or inorganic salts of iron, magnesium,copper and zinc-, together with pharmaceutically, nutraceutical orveterinary acceptable excipients and/or carriers.

This kit allows preparing in situ the synergistic combination of theinvention.

The invention also relates to pharmaceutical, nutraceutical orveterinary compositions comprising vitamins, amino acids, orpharmaceutically, nutraceutical or veterinary acceptable salts thereof;pharmaceutically, nutraceutical or veterinary acceptable organic orinorganic salts of iron, magnesium, copper and zinc; together withpharmaceutically, nutraceutical or veterinary acceptable excipientsand/or carriers; wherein the weight ratio of iron to copper is from 25:1to 900:1, of zinc to copper is from 30:1 to 400:1, and of magnesium tocopper is from 570:1 to 5800:1.

In a particular embodiment of these compositions, the weight ratio ofiron to copper is from 700:1 to 900:1, of zinc to copper is from 300:1to 400:1, and of magnesium to copper is from 2300:1 to 5800:1.

As will be depicted in the assays below, these compositions are bythemselves highly effective in terms of allowing proliferation of humanhair follicle papilla cells (HFDPC), human dermal fibroblasts (HDF) andhuman immortalized keratinocytes (HaCat). This feature makes thecompositions good for use as a medicament for skin treatment, inparticular for alleviating senescence processes of skin, and/or for skinwound healing. In addition, they are for use in the prevention and/ortreatment of mammal hair loss (in particular human hair loss) in amammal hair loss causing disease or disorder. They are also goodregenerating and reparation skin agents. Thus they can be used asingredients of cosmetic compositions.

Particular weight ratios of all iron, cooper, zinc and magnesium fromthe salts are, indicated as Fe/Cu/Zn/Mg: 868/1/307/2326-5730; and868/1/307/5730.

In a particular embodiment, the pharmaceutical, nutraceutical orveterinary compositions of the invention , comprise vitamin C, vitaminB1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B8, vitaminB9, glycine, methionine, phenylalanine, proline, cysteine, glutamicacid, zinc sulphate, copper sulphate, magnesium sulphate and ironsulphate together with pharmaceutically, nutraceutical or veterinaryacceptable excipients and/or carriers.

In another particular embodiment, the pharmaceutical, nutraceutical orveterinary compositions comprise protein hydrolysate, thiamine, niacin,pantothenic acid, pyridoxine, biotin, folic acid, riboflavin, ascorbicacid, citric acid, myo-inositol, calcium chloride, magnesium sulphate,potassium dihydrogenphosphate, zinc sulphate, copper sulphate, and ironethylendiaminetetraacetic chelate. More in particular, it comprisescasein hydrolysate.

These compositions, as well as the peptides of formula (I), areproliferation-inducing agents of mammal fibroblasts and of mammal dermalpapilla cells. Therefore, the invention lies in these areproliferation-inducing agents, selected from peptides of formula (I),and pharmaceutical, nutraceutical or veterinary compositions as definedabove, or mixtures thereof, for use in the prevention and/or treatmentof a hair loss in a hair loss causing disease.

The invention also relates to cell-free supernatants that previouslysupported the growth of a dedifferentiated plant cell suspensionculture, or a fraction of said cell-free supernatant, for use in theprevention and/or treatment of mammal hair loss in a mammal hair-losscausing disease, wherein said cell-free supernatant or said fractioncomprises peptides from 4 to 300 amino acids length, said peptidesselected from peptide plant growth factors, plant transcription factors,plant epigenetic factors and mixtures thereof, and said cell-freesupernatant or said fraction without having cytoplasmic cell contentsfrom the cell lysis and membranes and/or cell walls.

These supernatants without the inner content of cell cytoplasm andwithout the cell membranes and/or walls are herewith termed conditionednutrient media or conditioned media, or conditioned cell-medium. Thecell-free supernatants of the invention contain thus, not only amountsof those compounds initially present in the culture media wherededifferentiated plant cells were grown and that were not consumed, butalso other compounds that dedifferentiated plant cells released to themedia. It is thus proposed the use of these conditioned media withoutthe dedifferentiated cells, or of a fraction thereof comprising thesepeptide plant growth factors and/or transcription factors and/orepigenetic factors for prevention and/or treatment of mammal hair lossin a mammal hair loss causing disease, and more particularly hair lossin humans.

The fractions of said cell-free supernatants comprising peptides from 4to 300 amino acids length, being plant growth factors, plant epigeneticfactors or plant transcription factors, can also be defined as“isolated” compositions (or isolated fractions) comprising peptides from4 to 300 amino acids length and obtainable from the cell-freesupernatant that previously supported the growth of a dedifferentiatedplant cell suspension culture. In particular, isolation of suchcompositions comprise separating or recovering a liquid from the saidsupernatant with the peptide plant growth factors by means of at least aprotein separation process, said process comprising at least one ofchromatography (selected from solid-phase extraction (SPE),size-exclusion chromatography (SEC), and combination in cascadethereof), filtering (in particular by tangential flow filtration (TFF)),protein precipitation, and combinations thereof. These compositionscomprising peptide plant growth factors and/or plant transcriptionfactors so obtained are, in particular, for use as skin animal cellsrepairing and/or regenerating agents, and for use in the preventionand/or treatment of mammal hair loss in a mammal hair-loss causingdisease, as above exposed.

In a particular embodiment, the cell-free supernatant of adedifferentiated plant cell culture suspension, or a fraction thereofthat comprises peptides from 4 to 300 amino acids length for use in theprevention and/or treatment of mammal hair loss in a mammal hair-losscausing disease, is selected from a plant of the group consisting ofDaucus carota, Curcuma longa, Centella asiatica, Sarcocapnoscrassifolia, Vitis vinifera, Lithops pseudotruncatella, Morindacitrifolia, and Olea europaea.

More in particular the cell-free supernatant of a dedifferentiated plantcell culture suspension, or a fraction thereof that comprises peptidesfrom 4 to 300 amino acids length, for use in the prevention and/ortreatment of mammal hair loss in a mammal hair-loss causing disease isof Curcuma longa.

In a another particular embodiment, these cell-free supernatants orisolated fractions thereof include peptide cocktails, said cocktailscomprising at least a peptide plant growth factors selected from thegroup consisting of Phytosulphokine-α (PSK-α), Phytosulphokine-β(PSK-β), Plant Peptide Containing Sulphated Tyrosine-1 (PSY1), RapidAlkalinization Factor (RALF), Tracheary Element DifferentiationInhibitory Factor (TDIF), Clavata-3 (CLV3), Clavata-Embryo SurroundingRegion-Related (CLE), Tapetum Determinant-1 (TPD1), Epidermal PatterningFactor-1 (EPF1), Inflorescence Deficient in Abscission (IDA), EmbryoSurrounding Region-Related (ESR), Polaris peptide (PLS), Root meristemGrowth Factor (RGF), Egg Cell-Secreted Protein (EC1), C-terminallyEncoded peptide (CEP), Early Nodulin 40 (ENOD40), Systemin, S-locusCystein Rich proteins (SCR), and mixtures thereof, which means acombination of all of them, of only two, three, four or more than twountil comprising all of the listed peptides.

The cell-free supernatants or isolated fractions thereof include thepeptides of formula (I) of particular SEQ ID NO: 14 and 15. Thus, theyare also source of the active in hair loss treatment peptides of theinvention.

Other peptides than peptide plant growth factors are also present in aparticular embodiment of the cell-free supernatants that previouslysupported the growth of a dedifferentiated plant cell suspensionculture, or a fraction of said cell-free supernatants. These peptidesare in particular peptide plant transcription factors, including amongothers Wound-Induced Dedifferentiation (WIND), Wuschel (WUS), TeosinteBranched1/Cycloidea/Proliferating Cell Factor (TCP), and transcriptionalfactor for root meristem maintenance (PLETHORA), and mixtures thereof.These transcription factors regulate shoot meristem formation, stem cellmaintenance and somatic cell differentiation.

Traces of other compounds, usually known as epigenetic factors, are alsocomprised in the cell-free supernatants, or a fraction of said cell-freesupernatants due to the residual disruption of some cells duringculturing (as above enunciated), which can deliver the nuclear andcytoplasm contents in the culture media, although reduced to minimalamounts. Examples of these plant epigenetic factors are selected fromthe group consisting of Chromomethylase (CMT), Domains RearrangedMethyltransferase (DRM), Methyltransferase (MET), and some auxins,involved in the NA methylation processes; the chromatin remodellingfactor PICKLE of the CHP family (the CHD proteins derive their name fromthe presence of three domains of sequence similarity: a Chromatinorganization modifier domain (chromodomain), a SWI2/SNF2 ATPase/Helicasedomain, and a motif with sequence similarity to a DNA-binding domain),the chromatin remodelling factor DDM1 (from Decrease In DNAMethylation-1), involved in decondensation and remodelling of chromatin;the histone H3 methyltransferase Kryptonite (KYP), involved inremodelling of histones; small RNA molecules; and mixtures of all theseproteins and compounds. Epigenetic factors are to be understood as thosebiomolecules, particularly of peptide nature, that control marks in acell for the DNA controlling expression or silencing of genes.

Throughout the description and claims the word “comprise” and variationsof the word, are not intended to exclude other technical features,additives, components, or steps. Furthermore, the word “comprise”encompasses the case of “consisting of”. Additional objects, advantagesand features of the invention will become apparent to those skilled inthe art upon examination of the description or may be learned bypractice of the invention. The following examples are provided by way ofillustration, and they are not intended to be limiting of the presentinvention. Furthermore, the present invention covers all possiblecombinations of particular and preferred embodiments described herein.

EXAMPLES Example 1 Preparation of Peptides of Formula (I)

Assayed peptide sequences were assembled by solid phase methods using aFmoc-based protection scheme on a Rink amide resin. The side chain ofthe Tyr (Y) residues was protected with a t-butyl group. Deprotection ofthe N-terminal Fluorenylmethyloxycarbonyl chloride (Fmoc) groups wasdone with 20% piperidine in dimethylformamide (DMF) in 5 min. Synthesiswas performed in a Prelude automated synthesizer (Protein Technologies,Tucson, Ariz., USA). After chain assembly, the peptide-resin was treatedwith 90% trifluoroacetic acid-5% triisopropylsilane-5% water for 1 h,the corresponding filtrate was treated with 3 volumes of chilled ethylether to precipitate the peptide. After centrifugation, the supernatantwas carefully removed and he pellet (containing the peptides) wereredissolved in aqueous (5% v/v) acetic acid and lyophylized.Purification of the crude product was done by preparative reverse-phaseHPLC using water/acetonitrile gradients (both with 0.05% trifluoroaceticacid). The purified peptides were homogeneous (>95%) by HPLC and had theexpected molecular mass by electrospray mass spectrometry.

With this methodology the following sequences were obtained and assayedas will be illustrated in the next assays: CH₃—C(O)-YIYT-NH₂ (SEQ ID NO:6), CH₃—C(O)-YIYTQ-NH₂ (SEQ ID NO: 7), YIYT (SEQ ID NO: 12), YIYTQ (SEQID NO: 13), CH₃—C(O)-YIYT (SEQ ID NO: 16), CH₃—C(O)-YIYTQ (SEQ ID NO:17), YIYT-NH₂ (SEQ ID NO:18). and YIYTQ-NH₂ (SEQ ID NO: 19).

For in vivo assays (see below) acetyl tetrapeptide (SEQ ID NO: 6) wasdissolved (0.025% w/w) in a buffered solution containing salts, vitaminsand aminoacids. This buffered solution was as follows:

-   -   500 ml/L of a fortifying solution at 2×    -    Prepared from (a) a 100× solution, 30 ml/L [wherein the 100×        fortifying solution comprised ZnSO4.5H2O (860 mg/L); 100 μl        CuSO4.5H2O solution (25 g of CuSO4.5H2O per liter of Fe        solution), MgSO4.5H2O (18054 mg/L), Vitamine C (5000 mg/L),        Vitamin B1 (thiamine, 50 mg/L), vitamin B3 (niacin, 50 mg/L),        vitamin B5 (D-panthothenate calcium, 50 mg/L), vitamin B6        (pyridoxine, 50 mg/L), glycine (2000 mg/L), methionine (3000        mg/L), phenylalanine (1500 mg/L), proline (3000 mg/L), cysteine        (3000 mg/L), glutamic acid (750 mg/L), q.s. of FeSO4.7H2O        solution (Na2EDTA.2H2O (4.46 g/L), FeSO4.7H2O (3.503 g/L) in        miliQ water];    -    (b) vitamin stock solution 5×, 400 ml/L [wherein the vitamin        stick solution 5× is vitamin B2 (riboflavin, 2.5 mg/L), biotin        2.5 mg/L, vitamin B9 (folic acid, 2.5 mg/L) in miliQ water;    -    (c) an amino acid stock solution 20×, 100 ml/L [wherein the 20×        is glutamic acid (450 mg/L), L-phenilalanine (300 mg/L) in miliQ        water]    -    (d) Preservative 1%, 10 ml/L    -    (e) in 1 L of miliQ water.    -   500 ml/L of phosphate buffered saline (PBS) 2× at pH 6.8

For in vitro assays on hair follicle dermal papilla cells (HFDPC), humandermal fibroblasts (HDF), or immortalized epidermal keratinocytes(HaCat), synthesized peptides (SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQID NO: 19.) were dissolved in the culture media. The HFDPC were fromnormal human hair follicles from temporal scalp from a single donor: 54years old caucasian male were used (Ref. 602-05a. Lot. 2092. of CELLAPPLICATIONS, INC). The HDF were obtained from were obtained fromforeskin samples, surpluses from surgery of young donors (0-3 years old)and stablished by using the standard method of explants growth andenzymic dissociation of proliferating cells. And the immnortalized humankeratinocytes (HaCat) were obtained from aneuploid immortal keratinocytecell line from adult human skin

Example 2 Preparation of Cellfree Supernatants of Curcuma longa and ofFormulations Comprising Them for In Vivo Assays

C. longa Conditioned Media

It consisted of the extracellular media of a Curcuma longa cellsuspension mixed with water and/or glycerol (1:1).

The process for obtaining cell-free Curcuma longa cell suspension wascarried out by:

Equal volumes (1:1) of the extracellular media of a Curcuma longa cellculture at 1° brix were mixed with a fortifying solution containingminerals (inorganic salts), vitamins and amino acids. Brix degreesmeasures the amount of dry solids dissolved in a sample. This solids canbe sugar, salts, etc. and is a mode of determining cell growth incultures. Both components (extracellular media of a Curcuma longa andfortifying solution) were prepared separately and then mixed as:

-   -   Curcuma longa cells were cultured in SunB media as a cell        suspension until the supernatant was 1° brix.    -   The suspension was filtered using a 0.80 μm monofilament        polyesther filter.    -   1% Geogard Ultra was added as a preservative to the        extracellular media recovered by filtration.    -   Magnetic stirring was needed to properly incorporate the        preservative.    -   Equal volume of the fortifying solution (2×, as above) for        conditioned media was added to the extracellular media

Assay 1. In Vitro Proliferation of Human Fibroblasts of Hair FollicleDermal Papilla Cells (HFDPC)

In order to test the effects of the cell-free supernatants, as well asof the peptides of the invention, a proliferation assay was conductedonto human hair follicle papilla cells (CELL APPLICATIONS, INC; Normalhuman hair follicles from temporal scalp. Single donor: 54 years oldcaucasian male; Ref. 602-05a. Lot. 2092.).

Next Table 1 shows tested material and controls in FIG. 1 :

Assayed samples in FIG. 1 Basal control (Ctrl): non-treated cellsmaintained in culture media Positive control: cells treated withfibroblast growing factor (FGF) Positive control: cells treated withvascular endothelial growing factor (VEGF) Minoxidil (Mx) ((≥99% (TLC)),SIGMA, Ref. M4145. Stock solution: 16.67 mg/ml in Ethanol (25 mgMinoxidil + 1.5 ml Ethanol). Centella asiatica (CA) 3.0 μg/ml C1 and 6.0μg/ml C2 Curcuma longa (CL): 3.0 μg/ml C1 and 6.0 μg/ml C2 Daucus carota(DC): 1.6 μg/ml C1 and 1.5 μg/ml C2 (at 48 h) Vitis vinifera (W): 6.3μg/ml C1 and 12.5 μg/ml C2 Sarcocapnos crassifolia (SC): 12.5 μg/ml C1and 25.0 μg/ml C2 Morinda citrifolia (MC): 3.1 μg/ml C1 and 6.0 μg/ml C2Olea europaea (OE): 25.0 μg/ml C1 and 50.0 μg/ml C2 Lithops Sp. (LP):3.0 μg/ml C1 and 1.5 μg/ml C2 Peptide 4Aa (CH₃—C(O)—YIYT—NH₂) (SEQ IDNO: 6): 5.0 μg/ml C1 and 2.5 μg/ml C2 Peptide 5Aa (CH₃—C(O)—YIYTQ—NH₂)(SEQ ID NO: 7): 0.04 μg/ml C1 and 0.2 μg/ml C2

Next Table 2 shows the evaluated concentrations of peptides in FIGS. 2and 3 :

Peptide C1 (μg/ml) C2 (μg/ml) C3 (μg/ml) P4Aa 5.83 2.93 1.46 P5Aa 5.862.93 1.46

The culture media was a Papilla Cell Basal Medium supplemented with theGrowth Supplements kit (Relative composition: Fetal bovine serum, growthfactors and antibiotics. Application: 6% v/v).

The cell-free supernatants (also labelled as Pre-pre-LyoP3) wereobtained in particular as follows:

Cell suspension cultures of tested species were obtained from inoculumsof cell suspensions grown at 200 ml. The inoculums (⅕ of inoculum inrelation to the total volume of culture) were inoculated into 2000-mlflasks containing 800 ml of Murashige & Skoog (MS) liquid mediumsupplemented with 20-30 g/L of sucrose (for example, 30 g), and hormonesand placed in a rotary shaker at 100 rpm in the dark at 25° C. Cellcultures were grown for 12 days, when they were clarified (i.e. thecells were removed avoiding lysis) by centrifugation at 4600 rpm for 30min, to obtain the conditioned media, which was further studied. Inorder to better purify the conditioned media, a TFF (Tangencial FlowFiltration) was done in the resulting product. The process comprised; aflux step, an equilibration, a filtration, a diafiltration and a finalsanitization step. During the process, 2.5 l of PBS, 2.5 l of 20%Ethanol and NaOh 1N were used. After that TFF, a Reverse phasepurification (RP) was done (Oligo purification) with the aim ofrecovering those compounds secreted by plant cells during growing insuspension. The purification included 9 steps: addition of 5 Columnvolume (CV) water trifluoroacetic acid (TFA) 0.1%, addition of 5 CV CANTFA 0.1%, inclusion of 5 CV water TFA 0.1% and the addition of TFA toachieve 0.1%. Then the sample was loaded, collected and washed with a 1CV water TFA 0.1%. The elution was fractionated in 25-50-5-100% CAN withTFA 0.1%. The product was evaporated and resuspended in RP A. At the endof this purification pre-conditioned media 2 (pre cell-free supernatant)was obtained. This pre-conditioned media 2 was purified using a CEX(Cation exchange chromatography) and a AEX (Emphaze hybrid purifier).The process comprised 16 steps; addition of 5CV CEX A (sodium acetate 50mM pH 4.5), 5 CV CEX B (Sodium acetate 50 mM NaCl 1M pH 4.5) and 5CV CEXA. A dilution 1:5 and a pH adjust to 4.5 with CEX A was also done such asample loading, a collection of the sample, a washing with 1 CV CEX Aand a fractioned elution with CEX B at 125 mM-250 mM-500 mM-1M. Theelution received: 5 CV AEX A (Glycine 50 mM pH 9), 5 CV AEX B (Glycine50 mM NaCl 1 M at a pH 9) and 5 CV AEX A. A dilution 1:5 in AEX A and anadjust to pH 9 was done, as well as a sample loading, a collection ofthe sample, a washing with 1 CV AEX A and a fractioned elution with AEXB at 125 mM-250 mM-500 mM-1M. A final cell-free suspension (labeled Prepre-LyoP 3) was obtained. It was then lyophilized and resuspended foruse at the desired concentrations.

Materials

RP A: Water TFA 0.1%

RP B: CAN TFA 0.1%

CEX A: Sodium acetate 50 mM pH 4.5

CEX B: Sodium acetate 50 mM pH 4.5

AEX A: Glycine 50 mM pH 9

AEX B: Glycine 50 mM NaCl 1M pH 9

Assayed peptides were those synthesized as in Example 1.

HFDPC proliferation was assessed determining the proliferation index(%). Cell DNA replication was quantified by means of incorporatedbromodeoxiuridine (BrdU) into DNA of treated cells. BrdU assay allowsmeasuring cell proliferation based on cell capability of incorporatingBrdU during S-phase of the cell cycle. Cells being divided incorporatedBrdU that is further detected by means of antibodies andimmunocytochemistry detection.

Cells were seeded at confluence in a 96-well plate. After stabilizationand synchronization of cell cultures the tested products were added (atthe final concentration indicated in Table 1 of Examples. After that,BrdU was added to cultures and HFDPC were incubated at 37° C. untilcomplete BrdU incorporation. BrdU amounts were proportional to thenumber of cell divisions and thus to the growth of the treated culture.

Data are depicted in FIG. 1 , FIG. 2 and FIG. 3 , wherein it is shownthe proliferation index (%) of HFDPC, calculated as the percentage ofgrowing in relation to the basal control (non-treated HFDPC) which isaccorded a 100% value of proliferation index.

As can be seen in FIG. 1 , cell-free supernatants of the inventionpromoted cell proliferation taking as reference or basal control thenon-treated cells cultured with media. For some species, the cell-freesupernatants comprising the peptides with a molecular weight equal orlower than 30 kDa (from 4 to 300 amino acids length) the proliferationindex was much higher even than positive controls. If not, proliferationindex was of the same order than the positive controls. Furthermoretested peptide of SEQ ID NO: 6 showed the best results. Thus, confirmingthat peptides, as defined in the invention, are useful in the treatmentof mammal (in particular human) hair loss.

As can be observed in both FIGS. 2 and 3 , the peptides 4Aa and 5Aa canpromote cell proliferation independently of whether modifications arepresent in their end termini (columns 2-4) or not (column 1).

In order to determine the presence of the active peptides in the assayedcell-free supernatants, an HPLC chromatography was performed of thecell-free supernatant of Daucus Carota, Curcuma longa and Centellaasiatica (prepared as indicated in Assay 1) and of a peptide of SEQ IDNO: 6. HPLC was performed as follows:

The powder of lyophilized cell cultures of Centella asiatica, Daucuscarota and Curuma longa were resuspended in 2 mL of Water (0.1% NaOH1M), vortex and centrifuged at 10000 rcf during 5 min. Then thesupernatant was filtered through a 0.45 cellulose filter and injected atthe HPLC. The HPLC system consisted in a Waters 1795 chromatograph, aWaters Spherisorb® 5 μM OLDS2 4.6 μM×250 mm column and DAD detectorWaters 2996. The mobile phase consisted of Water+TFA 0.045% (A) andAcetonitrile+TFA 0.036% (B) at the following gradient (t (min), % B):(0, 0), (4, 0), (19, 100), (23, 0), (25, 0). The injection volume was 20μL, the flow rate was 1 mL/min and the wavelength was set at set at 220nm. Retention time of the peptide was 12.2 min.

From 12.0 to 12.5 minutes of elution (in particular at 12.165) theDaucus carota sample presented a peak as in the peptide chromatogram.These data are depicted in FIG. 4 . Due to the natural source of thepeptide detected in Daucus carota, derived from PSK-beta, the peptidesequence was that of SEQ ID NO: 14.

Although data not shown, SDS PAGE gels of the cell-free supernatants ofCurcuma longa and Centella asiatica showed protein bands at 100 KDa, ≈37KDa, ≈20 KDa, under 15 KDa and at 2 KDa for Centella asiatica. ForCurcuma longa there were also observed bands at 100 KDa, ≈25 KDa, andunder 15 KDa. The bands a under 15 KDa and more in particular in theband at 2 kDa contain the peptides of formula (I) in any of itssulphated or non-sulphate options.

Secretomas (supernatants that could be recovered from the HFDPC cultureschallenged with the tested materials of Table 1) were analysed fordetecting the presence and levels of animal cell growth factors and ofmicroRNAs associated with hair growth in dermal papilla. Although datanot shown, levels of insulin growth factor 1 (IGF-1) determined at 24 hand 48 h after treatment with the compounds of Table 1 were higher thanin non-treated HFDPCs and comparable or even higher than minoxidiltreated cells. These higher levels were meaningful when HFDPC weretreated with the peptide of SEQ ID NO: 6. Similar results (at 24 and 48h) were observed when analysing the levels of fibroblast growth factor 7(FGF-7). Both growth factors are associated with the promotion of hairgrowth.

From these secretomes of HFDPC, also the levels of the microRNAsmiRNA-31 and miRNA-22 were detected (FIG. 5 ). The peptide of SEQ ID NO:6, as well as the cell-free supernatant of Curcuma longa (CL) showed anincrease of the expression index of miRNA-31 (FIG. 5 (C) for the peptideat three different concentrations and (A) for Curcuma longa cell-freesupernatant at two different concentrations), in relation to thecontrols. Besides, the cell-free supernatant of Curcuma longa at twodifferent concentrations also showed a decrease of the expression indexof miRNA-22 (FIG. 5 (B)), in relation to the controls. miRNA-31 isassociated with the promotion of hair growth, and miRNA-22 with a thepromotion of hair loss (hair fall for the hair renewal).

Assay 2. In Vitro Proliferation of Human Dermal Fibroblasts (HDF)

In order to further test the effects of the peptides of the invention inan additional setting, a proliferation assay was conducted onto humandermal fibroblasts (HDF).

Cell Model:

Normal Human Dermal Fibroblasts (HDF) were obtained from foreskinsamples, surpluses from surgery of young donors (0-3 years old) andstablished by using the standard method of explants growth and enzymicdissociation of proliferating cells. Cells were propagated and grown inGrowth Medium (GM): Dulbecco's 1 g/L glucose medium, supplemented with10% foetal bovine serum (FBS, PAA); 2 mM L-glutamine (Lonza); andantibiotics (100 μg/ml Penicillin and 100 U/ml of Streptomycin, Lonza).For routine subcultivation and propagation of the primary culture, cellswere washed twice with PBS (Phosphate Phosphate buffered saline, pH7.4), harvested with trypsin—EDTA (Gibco) and counted in Neubauerchamber before its seeding in a new cell culture flask (Falcon, 75 cm2).

Assayed peptides were those synthesized as in Example 1.

Test products were prepared at the defined final concentrations by itsdilution in Maintenance Medium (Dulbecco's 1 g/L glucose medium,supplemented with 1% foetal bovine serum (FBS, PAA); 2 mM L-glutamine(Lonza); and antibiotics (100 μg/ml Penicillin and 100 U/ml ofStreptomycin, Lonza), just before each application.

Next Table 3 shows the evaluated concentrations of FIGS. 6 and 7 , foreach peptide of formula (I):

Peptide C1 (μg/ml) C2 (μg/ml) C3 (μg/ml) P4Aa 25 12.5 1.25 P5Aa 1.250.63 0.25

In FIG. 6 , the first column corresponds to the 4Aa peptide wherein bothends are free (SEQ ID NO:12); the second column corresponds to the 4Aapeptide wherein the N-terminal end is acetylated the C-terminal end isfree (SEQ ID NO:16); the third column corresponds to the 4Aa peptidewherein the N-terminal end is free and the C-terminal end is amidated(SEQ ID NO:18); and the forth column corresponds to the 4Aa peptidewherein the N-terminal end is acetylated and the C-terminal end isamidated (SEQ ID NO: 6).

In FIG. 7 , the first column corresponds to the 5Aa peptide wherein bothends are free (SEQ ID NO:13); the second column corresponds to the 5Aapeptide wherein the N-terminal end is acetylated the C-terminal end isfree (SEQ ID NO:17); the third column corresponds to the 5Aa peptidewherein the N-terminal end is free and the C-terminal end is amidated(SEQ ID NO:19); and the forth column corresponds to the 5Aa peptidewherein the N-terminal end is acetylated and the C-terminal end isamidated (SEQ ID NO: 7).

HFD proliferation was assessed determining the proliferation index (%).Cell DNA replication was quantified by means of incorporatedbromodeoxiuridine (BrdU) into DNA of treated cells. BrdU assay allowsmeasuring cell proliferation based on cell capability of incorporatingBrdU during S-phase of the cell cycle. Cells being divided incorporatedBrdU that is further detected by means of antibodies andimmunocytochemistry detection.

Cells were seeded at confluence in a 96-well plate. After stabilizationand synchronization of cell cultures the tested products were added.After that, BrdU was added to cultures and HFD were incubated at 37° C.until complete BrdU incorporation. BrdU amounts were proportional to thenumber of cell divisions and thus to the growth of the treated culture.

Data are depicted in FIG. 6 and FIG. 7 , wherein it is shown theproliferation index (%) of HDF, calculated as the percentage of growingin relation to the basal control (non-treated HDF) which is accorded a100% value of proliferation index.

As it was observed in experiments performed on HFDPC, FIGS. 6 and 7reveal that both 4Aa and 5Aa peptides can promote cell proliferation ofHDF independently of whether modifications are present in their endtermini (columns 2-4) or not (column 1). In the case of the 4Aa peptide,FIG. 6 shows that the presence of at least one modification furtherincreases it cell proliferation-promoting activity.

Assay 3. In Vivo Test of Peptides of Formula (I) and of Cell-FreeSupernatants of Curcuma longa

Curcuma longa cell-free supernatant prepared as indicated in Example 2,as well as the peptide of SEQ ID NO: 6 in the PBS buffered fortifyingsolution 2× disclosed in Example 1 were tested in males and females(n=60, from 18 to 60 years old) with hair-loss problems. There wereexcluded those volunteers that received a capillary treatment threemonths before the assay, volunteers with cutanic pathologies at scalp(psoriasis, dermatitis, etc.) and allergy to compounds in theformulations.

Tested compounds were daily applied.

At time 0 (T0) there were taken scalp pictures (frontal and occipitalparts), trichograms for determining the hair phase cycle (telogen oranagen phase), the combing test (number of hair lost during combing),and capillary density by microphotography with Trichoscan®.

At t=45 days and t=90 days, there were performed the combing test andthe density tests (Trichoscan®)

At t=150 days again as in t=0.

Volunteers were separated in three groups. Group 1 receiving SEQ ID NO:6 in the PBS buffered fortifying solution 2× disclosed in Example 1;Group 2 receiving Curcuma longa cell-free supernatant prepared asindicated in Example 2, and Group 3 the placebo.

Data are depicted in next Table 2 for capillary density. This tableincludes the average values determined of capillary density increaseafter evaluating in each volunteer the number of hairs in a 0.25 cm²are, capillary density at t=0 and t=90, and said increase in relation tot=0 value

TABLE 4 Increase in capillary density Group Capillary density increase 1(volunteers 1 to 20, males and females) 14.66% 2 (volunteers 21 to 40,males and females) 15.61% 3 (volunteers 41 to 60, males and females)2.80%

In Table 5, more detailed results of hair density and hair densityincrease of volunteers of group 1:

TABLE 5 Nr HAIRS/ HAIR HAIR DENSITY HAIR DENSITY 0.25 cm2 DENSITY 1/cm2INCREASE INCREASE Voluntary T0 T90 T150 T0 T90 T150 T90 T150  1 31 31 31123.31 123.31 123.31  0.00%  0.00%  2 15 21 26  59.67  83.53 103.4239.99% 73.32%(*)  3 31 34 34 123.31 135.24 135.24  9.67%  9.67%  4 26 2937 103.42 115.35 147.18 11.54% 42.31%  5 24 28 30  95.47 111.38 119.3316.66% 24.99%  6 33 35 35 131.27 139.22 139.22  6.06%  6.06%  7 22 31 30 87.51 123.31 119.33 40.91% 36.36%  8 27 30 31 107.4 119.33 123.1111.11% 14.63%  9 22 27 26  87.51 107.4 103.42 22.73% 18.18% 10 32 43 39127.29 171.04 155.13 34.37% 21.87% 11 37 39 35 147.18 155.13 139.22 5.40% −5.41% 12 35 41 36 139.22 163.09 143.2 17.15%  2.86% 13 28 30 38111.38 119.33 151.15  7.14% 35.71% 14 29 29 29 115.35 115.35 115.35 0.00%  0.00% 15 27 30 31 107.4 119.33 123.31 11.11% 14.81% 16 52 47 48206.84 186.95 190.93 −9.62%(*) −7.69%(*) 17 31 — — 123.31 — — — — 18 2034 32  79.55 135.24 127.29 70.01%(*) 60.01% 19 21 22 29  83.53  87.51115.35  4.76% 38.09% 20 27 27 29 107.4 107.4 115.35  0.00%  7.40% Mean:Mean: 14.03% 19.27% (*)minimal and maximal values, not considered whendetermining mean of values

In Table 6, more detailed results of hair density and hair densityincrease of volunteers of group 2:

TABLE 6 Nr HAIRS/ HAIR HAIR DENSITY HAIR DENSITY 0.25 cm2 DENSITY 1/cm2INCREASE INCREASE Voluntary T0 T90 T150 T0 T90 T150 T90 T150  1 38 45 39151.15 179 155.13  18.43%  2.63%  2 33 33 33 131.27 131.27 131.27  0.00% 0.00%  3 29 43 54 115.35 171.04 214.8  48.28% 86.22%(*)  4 26 29 30103.42 115.35 119.33  11.54% 15.38%  5 26 39 39 103.42 155.13 155.13 50.00% 50.00%  6 38 39 42 151.15 155.13 167.06  2.63% 10.53%  7 23 3735  91.49 147.18 139.22  60.87%(*) 52.17%  8 21 26 27  83.53 103.42107.4  23.81% 28.58%  9 33 39 43 131.27 155.13 171.04  18.18% 30.30% 1031 — — 123.31 — — — — 11 36 40 41 143.2 159.11 163.09  11.11% 13.89% 1224 28 27  95.47 111.38 107.4  16.66% 12.50% 13 29 27 30 111.35 107.4119.33  3.55%  7.17% 14 19 23 19  75.58  91.49  75.58  21.05%  0.00% 1536 37 34 143.2 147.8 135.24  3.21% −5.56%(*) 16 63 70 61 250.6 278.44242.64  11.11% −3.18% 17 30 31 32 119.33 123.31 127.29  3.34%  6.67% 1834 35 34 135.24 139.22 135.24  2.94%  0.00% 19 28 34 42 111.38 135.24167.06  21.42% 49.99% 20 22 19 26  87.57  75.58 103.42 −13.69%(*) 18.10%Mean: Mean:  15.30% 17.34% (*):minimal and maximal values, notconsidered when determining mean of values

Scalp pictures (frontal and occipital parts), and capillary density bymicrophotography with Trichoscan® are depicted for a volunteer in group1 (FIG. 8 ) and for a volunteer in group 2 (FIG. 9 ). In both figures(A) and (B) show pictures of occipital and frontal parts, respectively.In (C) the Trichoscan® images.

From all assays on hair density it could be concluded that with a hairdensity increase of 19.27% (group 1) up to new 14000 hairs appeared inthe whole hair. In the same way, with a hair density increase of 17.34%(group 2), up to new 13500 hairs appeared in the whole hair.

Next Table 7 shows the combing test results. In particular, this tableincludes the average values determined for combing test (number of hairlost during combing). From the combing test results at t=0, t=45 andt=90 the percentage of reduction of hair loss is calculated contrastingvalues of t=0 with the values at t=45 and at t=90.

TABLE 7 % of reduction of hair loss % hair-loss reduction t = 0 vs t = 0vs Group t = 45 t = 90 1 (volunteers 1 to 20, males and females) 40.4839.72 2 (volunteers 21 to 40, males and females) 52.94 56.79 3(volunteers 41 to 60, males and females) 23.44 24.22

In Table 8, more detailed results of hair loss of volunteers of group 1:

TABLE 8 % REDUCTION % REDUCTION % REDUCTION COMBING COMBING OF HAIR LOSSCOMBING OF HAIR LOSS COMBING OF HAIR LOSS Voluntary TEST T0 TEST T45 T0VS. T45 TEST T90 T0 VS. T90 TEST T150 T0 vs. T150  1  31  24 −22.58  23−25.81  20 −35.48(*)  2  39  14 −64.10  22 −43.59  16 −58.97  3  80  46−42.50  59 −26.25  36 −55.00  4  69  92  33.33  62 −10.14  20 −71.01  5 51  7 −86.27  6 −88.24  5 −90.20  6 122  54 −55.74  88 −27.87  13−89.34  7  43  23 −46.51  28 −34.88  13 −69.77  8  42  13 −69.05  28−33.33  19 −54.76  9  51  88  72.55  36 −29.41  22 −56.86 10  65 184183.08(*) 154 136.92(*)  24 −63.08 11  60  34 −43.33  60  0.00  18−70.00 12 104  58 −44.23  50 −51.92  12 −88.46 13 115  60 −47.83  61−46.96  20 −82.61 14 286  62 −78.32  84 −70.63  23 −91.96(*) 15  72  50−30.56  27 −62.50  29 −59.72 16 535  10 −98.13(*)  12 −97.76(*)  95−82.24 17  49  31 −36.73 — — — — 18  89  28 −68.54  45 −49.44  19 −78.6519 147 101 −31.29 118 −19.73  42 −71.43 20 200  66 −67.00  91 −54.50 167−16.50 Mean Mean Mean  40.48  39.72  68.15 (*)minimal and maximalvalues, not considered when determining mean of values

In Table 9, more detailed results of hair loss of volunteers of group 2:

TABLE 9 % REDUCTION % REDUCTION % REDUCTION COMBING COMBING OF HAIR LOSSCOMBING OF HAIR LOSS COMBING OF HAIR LOSS Voluntary TEST T0 TEST T45 T0vs T45 TEST T90 T0 vs. T90 TEST T150 T0 vs. T150 21  38  46  21.05(*) 90136.84(*)  35  −7.89 22  41  11 −73.17 20 −51.22  37  −9.76 23 160  12−92.50(*) 53 −66.88  26 −83.75 24 108  19 −82.41 31 −71.30  28 −74.07 25 83  75  −9.64 61 −26.51  51 −38.55 26  82  12 −85.37 18 −78.05  9−89.02 27 107  19 −82.24 11 −89.72(*)  16 −85.05 28  39  33 −15.38 21−46.15  31 −20.51 29  86  35 −59.30 46 −46.51  38 −55.81 30  67 — — — —— — 31 122  24 −80.33 19 −84.43  4 −96.72(*) 32 142 121 −14.79 99 −30.28167  17.61(*) 33 110  21 −80.91 22 −80.00  17 −84.55 34  59  27 −54.2421 −64.41  55  −6.78 35  54  12 −77.78 19 −64.81  23 −57.41 36 105  64−39.05 45 −57.14  26 −75.24 37  58  58  0.00 36 −37.93  14 −75.86 38  92 66 −28.26 63 −31.52  43 −53.26 39  58  23 −60.34 33 −43.10  20 −65.5240  81  35 −56.79 12.00 −85.19  19.00 −76.54 Mean Mean Mean  52.94 56.79  56.45 (*)minimal and maximal values, not considered whendetermining mean of values

As derivable from Tables 7 to 9, a meaningful reduction of the % of hairloss was observed when the peptide of SEQ ID NO: 6 or the Curcuma longaconditioned media were applied to volunteers. Comparing results at T45,T90 and T150 with t=0 (T0) the percentage of reduction of hair loss wasalready worthy at T45 and increased with time for the peptide, andmaintained after T45 for the Curcuma longa.

Assay 4. In Vitro Proliferation Assay to Test the Effect of FortifyingSolutions.

In order to test the effect of a fortifying solution alone,proliferation assays were conducted onto human hair follicle papillacells (HFDPC), human dermal fibroblasts (HDF) and human immortalizedkeratinocytes (HaCat).

This fortifying solution is also termed as buffered solution, nutritivesolution, or generically as a pharmaceutical, nutraceutical orveterinary composition comprising vitamins, amino acids, andpharmaceutically, nutraceutical or veterinary acceptable salts of iron,magnesium, copper and zinc, together with pharmaceutically,nutraceutical or veterinary acceptable excipients and/or carriers,

Thus, one of the buffered solutions assayed was as follows:

-   -   500 ml/L of a fortifying solution at 2×    -    Prepared from (a) a 100× solution, 30 ml/L [wherein the 100×        fortifying solution comprised ZnSO4.5H2O (860 mg/L); 100 μl        CuSO4.5H2O solution (25 g of CuSO4.5H2O per liter of Fe        solution), MgSO4.5H2O (18054 mg/L), Vitamine C (5000 mg/L),        Vitamin B1 (thiamine, 50 mg/L), vitamin B3 (niacin, 50 mg/L),        vitamin B5 (D-panthothenate calcium, 50 mg/L), vitamin B6        (pyridoxine, 50 mg/L), glycine (2000 mg/L), methionine (3000        mg/L), phenylalanine (1500 mg/L), proline (3000 mg/L), cysteine        (3000 mg/L), glutamic acid (750 mg/L), q.s. of FeSO4.7H2O        solution (Na2EDTA.2H2O (4.46 g/L), FeSO4.7H2O (3.503 g/L) in        miliQ water];    -    (b) vitamin stock solution 5×, 400 ml/L [wherein the vitamin        stick solution 5× is vitamin B2 (riboflavin, 2.5 mg/L), biotin        2.5 mg/L, vitamin B9 (folic acid, 2.5 mg/L) in miliQ water;    -    (c) an amino acid stock solution 20×, 100 ml/L [wherein the 20×        is glutamic acid (450 mg/L), L-phenilalanine (300 mg/L) in miliQ        water]    -    (d) Preservative 1%, 10 ml/L    -    (e) in 1 L of miliQ water.    -   500 ml/L of phosphate buffered saline (PBS) 2× at pH 6.8 HFDPC,        HDF, and the methodology of the assay have been described in the        Assays 1 and 2. Human immortalized keratinocytes (HaCat) were        obtained from aneuploid immortal keratinocyte cell line from        adult human skin . . .

Data are depicted in FIGS. 10, 11 and 12 , wherein it is shown therelative cell viability of HFDPC, HDF, and keratinocytes, respectively.The relative cell viability is calculated as the percentage of livingcells in relation to the basal control (non-treated cells) which isaccorded a 100% value of cell viability.

As observed in the FIG. 10-12 , the fortifying solution of the inventionpromotes the proliferation of the three different cell types, taking asreference or basal control the non-treated cells cultured with media.

It was tested another solution comprising vitamins, amino acids, andpharmaceutically, nutraceutical or veterinary acceptable salts of iron,magnesium, copper and zinc, together with pharmaceutically,nutraceutical or veterinary acceptable excipients and/or carriers. Inparticular, a composition comprising protein hydrolysate (caseinhydrolysate), thiamine, niacin, pantothenic acid, pyridoxine, biotin,folic acid, riboflavin, ascorbic acid, citric acid, myo-inositol,calcium chloride, magnesium sulphate, potassium phosphate, zincsulphate, copper sulphate, and iron chelate was tested.

Data are depicted in FIG. 13-15 , wherein it is shown the relative cellviability of HFDPC, HDF, and keratinocytes, respectively.

FIG. 13-15 show that this alternative solution also promotes theproliferation of the three different cell types tested, as compared tothe non-treated cells.

Both compositions assayed comprised salts of iron, copper, zinc, andmagnesium, wherein the weight ratio of iron:copper:zinc :magnesium wasfrom ratio 25:1:30:570 to the ratio 900:1:400:5800. This means that bothcompositions assayed comprised salts of iron, copper, zinc, andmagnesium, wherein the weight ratio of iron to copper was from 25:1 to900:1, of zinc to copper was from 30:1 to 400:1, and of magnesium tocopper was from 570:1 to 5800:1.

In particular, the weight ratio of iron:copper:zinc:magnesium was868:1:307:5730 in the first solution (that of FIGS. 10-12 ), and of25:1:307:2856 in the second solution (of FIGS. 13-15 ) above described.These weight ratios in this particular examples corresponded to mg ofthe indicated element (Fe, Cu, Zn and Mg). More in particular theycorrespond to mg/L in the compositions.

Assay 5. In Vitro Proliferation Assay to Test the Effect of the Peptidesof Formula (I) Combined with the Fortifying Solution (Comprising Salts,Vitamins and Amino Acids) of the Invention

In order to test whether there was a synergistic effect of thecombination of the peptides of the invention and the solutionscomprising salts of iron, copper, magnesium and zinc, together withvitamins and amino acids of the invention, proliferation assays wereconducted onto human dermal fibroblasts (HDF).

The assayed peptide, the fortifying solution and the culture of HDF havebeen already described in the previous assays.

Next Table 10 shows tested material and controls:

Assayed samples Basal control (Ctrl): non-treated cells maintained inculture media Peptide 4Aa (CH₃—C(O)—YIYT—NH₂ ) (SEQ ID NO: 6) NutritiveSolution: is the fortifying solution as described in Assay 4 with theratio of iron:copper:zinc:magnesium was 868:1:307:5730. P + NS: Peptide4Aa and Nutritive Solution used in combination.

Data are depicted in FIG. 16 , wherein it is shown the proliferationindex (%) of HDF, calculated as the percentage of growing in relation tothe basal control (non-treated HDF) which is accorded a 100% value ofproliferation index.

FIG. 16 reveals that both the 4Aa peptide or the fortifying solutionalone can promote cell proliferation. Moreover, it is shown that the useof both in combination increases more than two-fold the proliferation ofHDF as compared to their use alone. Therefore, it is clear that the useof the peptides and the fortifying solution of the invention incombination elicits a synergistic effect on the proliferation of HDF.

Assay 6. Wound Healing Assay (Reference Example)

In order to test the effect of the sulfaction state of the peptides ontheir activity, it is herewith provided as reference example a woundhealing assay conducted onto human dermal fibroblasts (HDF). The aim ofthis example is to show that sulfated peptides have the same activity asthe non-sulfated peptides.

Next Table 11 shows features of tested products and of controls:

TABLE 11 Samples assayed in wound healing test Assayed samples Basalcontrol (BC) 0.1% Fetal bovine serum (FBS) Positive control (Ctrl+) 10%(FBS) Positive control (Ctrl+) tissular growing factor-β1 (TGF-β1)Peptide 4Aa (SEQ ID NO: 6): 5 μg/ml and 0.05 μg/ml Peptide 4AaS1 (SEQ IDNO: 2): 5 μg/ml and 0.05 μg/ml Peptide 4AaS2 (SEQ ID NO: 3): 5 μg/ml and0.05 μg/ml

Human dermal fibroblasts were seeded in a 24-well plate and were grownto confluence. A 2 mm width scratch was done on the culture monolayer.Then the test products in culture medium were added and thecicatrisation process was followed by means of phase contrastmicroscopy. With this aim, photographs were taken at initial of the test(T=0 h) before the scratching and after the treatment (at 12 h and at 72h). Cicatrisation process was evaluated by quantifying wound areareduction at each time.

Data at the end of the test (5 days of treatment) are depicted in FIG.17 , wherein the wound healing potential is depicted as the Healed Area(in μm²) for assayed peptide.

The results derive from a triplicate test. Values of cicatrised area (or% of cicatrisation) are the mean values.

From this FIG. 17 it is directly deduced that all tested peptides weremore effective than the basal control and they provided effects similarto the positive controls or even higher. This data allow affirming thatthe peptides are real wound healing promoters.

Furthermore, FIG. 17 reveals that the activity of 4Aa is independent ofits sulfaction state, since the 4AaS1 and 4AaS2 sulfated peptides havethe same activity as the 4Aa non-sulfated peptide.

REFERENCES CITED IN THE APPLICATION

-   -   U.S. Pat. No. 4,139,619    -   U.S. Pat. No. 4,596,812    -   U.S. Pat. No. 6,281,241    -   WO2007113851    -   WO2006087759    -   Pumthong et al. “Curcuma aeruginosa, a novel botanically derived        5α-reductase inhibitor in the treatment of male-pattern        baldness: a multicenter, randomized, double-blind,        placebo-controlled study”, Journal of Dermatological        Treatment.-2012; vol. no. 23, pp.: 385-392    -   Matsubayashi et al., “Phytosulfokine, sulphated peptides that        induce the proliferation of single mesophyll cells of Asparagus        officinalis L.”, Proc. Natl. Acad. Sci.- 1996, vol. no. 93, pp.:        7623-7627.

1. A peptide sequence of formula (I), or a pharmaceutically,nutraceutical or veterinary acceptable salt thereof,(R¹)_((m))-Xaa₁IXaa₂T(Q)_((p))(R²)_((n))   (I) (SEQ ID NO: 1) wherein:R¹ is a —C(O)—(C₁-C₂₀)-alkyl radical, acylating N-terminal residue ofthe peptide sequence; R² is a —NR₄R₅ radical, amidating C-terminalresidue of the peptide sequence, being R₄ and R₅ selected from H, and(C₁-C₃)-alkyl; m, n and p, are integers from 0 to 1; and Xaa₁ and Xaa₂are tyrosine residues with the side-chain hydroxyl group of the aminoacid optionally replaced by a radical —OSO₃R³; being R³ selected from Hand (C₁-C₃)-alkyl; for use in the prevention and/or treatment of mammalhair loss in a hair loss causing disease and/or disorder.
 2. The peptidefor use according to claim 1, that is selected from the group consistingof: CH₃—C(O)-Xaa₁IYT-NH₂ (SEQ ID NO: 2), wherein Xaa₁ is a tyrosineresidue with the side-chain hydroxyl group replaced by a radical —OSO₃H;CH₃—C(O)-YIXaa₂T-NH₂ (SEQ ID NO: 3), wherein Xaa₂ is a tyrosine residuewith the side-chain hydroxyl group replaced by a radical —OSO₃H;CH₃—C(O)-Xaa₁IYTQ-NH₂ (SEQ ID NO: 4) wherein Xaa₁ is a tyrosine residuewith the side-chain hydroxyl group replaced by a radical —OSO₃H;CH₃—C(O)-YIXaa₂TQ-NH₂ (SEQ ID NO: 5), wherein Xaa₂ is a tyrosine residuewith the side-chain hydroxyl group replaced by a radical —OSO₃H;CH₃—C(O)-YIYT-NH₂ (SEQ ID NO: 6); CH₃—C(O)-YIYTQ-NH₂ (SEQ ID NO: 7) Xaa₁IYT (SEQ ID NO: 8), wherein Xaa₁ is a tyrosine residue with theside-chain hydroxyl group replaced by a radical —OSO₃H; YIXaa₂T (SEQ IDNO: 9), wherein Xaa₂ is a tyrosine residue with the side-chain hydroxylgroup replaced by a radical —OSO₃H; Xaa₁IYTQ (SEQ ID NO: 10) whereinXaa₁ is a tyrosine residue with the side-chain hydroxyl group replacedby a radical —OSO₃H; YIXaa₂TQ (SEQ ID NO: 11), wherein Xaa₂ is atyrosine residue with the side-chain hydroxyl group replaced by aradical —OSO₃H; (SEQ ID NO: 12) YIYT; (SEQ ID NO: 13) YIYTQ;

Xaa₁IXaa₂T (SEQ ID NO: 14), wherein Xaa₁ and Xaa₂ are both a tyrosineresidue with the side-chain hydroxyl group replaced by a radical —OSO₃H;Xaa₁IXaa₂TQ (SEQ ID NO: 15), wherein Xaa₁ and Xaa₂ are both a tyrosineresidue with the side-chain hydroxyl group replaced by a radical —OSO₃H;(SEQ ID NO: 16) CH_(3—)C(O)- YIYT; (SEQ ID NO: 17) CH_(3—)C(O)- YIYTQ;(SEQ ID NO: 18) YIYT-NH₂; (SEQ ID NO: 19) YIYTQ-NH₂;

and mixtures thereof.
 3. The peptide for use according to claim 2,wherein the peptide is selected from the group consisting of:(SEQ ID NO: 6) CH₃—C(O)- YIYT-NH₂; (SEQ ID NO: 7)CH_(3—)C(O)- YIYTQ-NH₂;

Xaa₁IYT (SEQ ID NO: 8), wherein Xaa₁ is a tyrosine residue with theside-chain hydroxyl group replaced by a radical —OSO₃H; YIXaa₂T (SEQ IDNO: 9), wherein Xaa₂ is a tyrosine residue with the side-chain hydroxylgroup replaced by a radical —OSO₃H; (SEQ ID NO: 12) YIYT;(SEQ ID NO: 13) YIYTQ;

Xaa₁IXaa₂T (SEQ ID NO: 14), wherein Xaa₁ and Xaa₂ are both a tyrosineresidue with the side-chain hydroxyl group replaced by a radical —OSO₃H;Xaa₁IXaa₂TQ (SEQ ID NO: 15), wherein Xaa₁ and Xaa₂ are both a tyrosineresidue with the side-chain hydroxyl group replaced by a radical —OSO₃H;and mixtures thereof.
 4. A pharmaceutical, nutraceutical, or veterinarycomposition comprising a peptide of formula (I), or a pharmaceuticallyor veterinary acceptable salt thereof, together with pharmaceutically,nutraceutical or veterinary acceptable excipients and/or carriers foruse in the prevention and/or treatment of a hair loss in a hair losscausing disease.
 5. The pharmaceutical, nutraceutical or veterinarycomposition for use according to claim 4, wherein said compositionfurther comprises vitamins, amino acids, and pharmaceutically,nutraceutical or veterinary acceptable salts of iron, magnesium, copperand zinc.
 6. The pharmaceutical, nutraceutical or veterinary compositionfor use according to claim 5, wherein the vitamins are selected from thegroup consisting of vitamin C, vitamin B1, vitamin B3, vitamin B5,vitamin B6, vitamin B2, vitamin B8, vitamin B9, and mixtures thereof. 7.The pharmaceutical, nutraceutical or veterinary composition for useaccording to any of claims 5-6, wherein the amino acids are selectedfrom the group consisting of glutamic acid, phenylalanine, glycine,methionine, proline, cysteine and mixtures thereof.
 8. Thepharmaceutical, nutraceutical or veterinary composition for useaccording to any one of claims 5-7, wherein the weight ratio of iron tocopper is from 25:1 to 900:1, of zinc to copper is from 30:1 to 400:1,and of magnesium to copper is from 570:1 to 5800:1.
 9. Thepharmaceutical, nutraceutical or veterinary composition for useaccording to any of claims 5-8, wherein the pharmaceutically,nutraceutical or veterinary acceptable salts of iron, magnesium, copperand zinc are inorganic salts selected from the group consisting of ironsulphate, copper sulphate, zinc sulphate, magnesium sulphate, andmixtures thereof.
 10. The pharmaceutical, nutraceutical or veterinarycomposition for use according to any one of claims 4-9, comprising apeptide of formula (I), or a pharmaceutically or veterinary acceptablesalt thereof, vitamin C, vitamin B1, vitamin B2, vitamin B3, vitamin B5,vitamin B6, vitamin B8, vitamin B9, glycine, methionine, phenylalanine,proline, cysteine, glutamic acid, zinc sulphate, copper sulphate,magnesium sulphate and iron sulphate together with pharmaceutically,nutraceutical or veterinary acceptable excipients and/or carriers. 11.The pharmaceutical, nutraceutical or veterinary composition for useaccording to any of claims 4-10, wherein the peptide of formula (I) isprovided in the composition as an ingredient comprised in a cell-freesupernatant that previously supported the growth of a dedifferentiatedplant cell suspension culture, or a fraction of said cell-freesupernatant, wherein said cell-free supernatant or said fractioncomprises peptides from 4 to 300 amino acids length, said peptidesselected from peptide plant growth factors, plant transcription factors,epigenetic factors and mixtures thereof, and said cell-free supernatantor said fraction without having cytoplasmic cell contents from the celllysis and membranes and/or cell walls.
 12. The pharmaceutical,nutraceutical or veterinary composition for use according to claim 11,wherein the peptide of formula (I) is provided in the composition as aningredient of a cell-free supernatant of a dedifferentiated plant cellculture suspension from a plant selected from the group consisting ofDaucus carota, Centella asiatica, Sarcocapnos crassifolia, Curcumalonga, Vitis vinifera, Lithops pseudotruncatella, Morinda citrifolia,and Olea europaea.
 13. The peptide sequence of formula (I) or apharmaceutically, nutraceutical or veterinary acceptable salt thereoffor use according to any of claims 1-3, or the pharmaceutical,nutraceutical or veterinary composition for use according to any ofclaims 4-12, wherein the mammal hair loss causing disease is selectedfrom alopecia, hypotrichosis, vitamin or mineral deficiency,trichotillomania, hypothyroidism, tightly pulled hair, a scalp fungalinfection, and combinations thereof.
 14. A pharmaceutical, nutraceuticalor veterinary composition comprising vitamins, amino acids, orpharmaceutically, nutraceutical or veterinary acceptable salts thereof;pharmaceutically, nutraceutical or veterinary acceptable organic orinorganic salts of iron, magnesium, copper and zinc-, together withpharmaceutically, nutraceutical or veterinary acceptable excipientsand/or carriers, the composition further comprising a peptide of formula(I), or a pharmaceutically, nutraceutical or veterinary acceptable saltthereof; and/or a cell-free supernatant of a dedifferentiated plant cellculture suspension, or a fraction of said cell-free supernatant, whereinsaid cell-free supernatant or said fraction comprises peptides from 4 to300 amino acids length, said peptides selected from peptide plant growthfactors, plant transcription factors, epigenetic factors, and mixturesthereof, said peptide plant growth factors comprising the peptide offormula (I), and said cell-free supernatant or said fraction withouthaving cytoplasmic cell contents from the cell lysis and membranesand/or cell walls.
 15. The pharmaceutical or veterinary compositionaccording to claim 14, which is a topical composition comprisingvitamins, amino acids, or pharmaceutically, nutraceutical or veterinaryacceptable salts thereof; pharmaceutically or veterinary acceptablesalts of iron, magnesium, copper and zinc; together withpharmaceutically or veterinary acceptable excipients and/or carriers,the composition further comprising a peptide of formula (I), or apharmaceutically or veterinary acceptable salt thereof; and/or acell-free supernatant of a dedifferentiated plant cell culturesuspension, or a fraction of said cell-free supernatant, wherein saidcell-free supernatant or said fraction comprises peptides from 4 to 300amino acids length, said peptides selected from peptide plant growthfactors, plant transcription factors and mixtures thereof, said peptideplant growth factors comprising the peptide of formula (I), and saidcell-free supernatant or said fraction without having cytoplasmic cellcontents from the cell lysis and membranes and/or cell walls.
 16. Thepharmaceutical, nutraceutical or veterinary composition according to anyof claims 14-15, wherein the peptide of formula (I) is selected from:(SEQ ID NO: 6) CH₃—C(O)- YIYT-NH₂; (SEQ ID NO: 7)CH_(3—)C(O)- YIYTQ-NH₂;

Xaa₁ IYT (SEQ ID NO: 8), wherein Xaa₁ is a tyrosine residue with theside-chain hydroxyl group replaced by a radical —OSO₃H; YIXaa₂T (SEQ IDNO: 9), wherein Xaa₂ is a tyrosine residue with the side-chain hydroxylgroup replaced by a radical —OSO₃H; (SEQ ID NO: 12) YIYT;(SEQ ID NO: 13) YIYTQ;

Xaa₁IXaa₂T (SEQ ID NO: 14), wherein Xaa₁ and Xaa₂ are both a tyrosineresidue with the side-chain hydroxyl group replaced by a radical —OSO₃H;Xaa₁IXaa₂TQ (SEQ ID NO: 15), wherein Xaa₁ and Xaa₂ are both a tyrosineresidue with the side-chain hydroxyl group replaced by a radical —OSO₃H;CH₃—C(O)-YIYT (SEQ ID NO: 16); CH₃—C(O)-YIYTQ (SEQ ID NO: 17);(SEQ ID NO: 18) YIYT-NH₂; (SEQ ID NO: 19) YIYTQ-NH₂;

and mixtures thereof.
 17. The pharmaceutical, nutraceutical orveterinary composition according to any of claims 14-16, comprising thepeptide of formula (I), or a pharmaceutically, nutraceutical orveterinary acceptable salt thereof; vitamins, amino acids, andpharmaceutically, nutraceutical or veterinary acceptable salts of iron,magnesium, copper and zinc, wherein the weight ratio of iron to copperis from 25:1 to 900:1, of zinc to copper is from 30:1 to 400:1, and ofmagnesium to copper is from 570:1 to 5800:1.
 18. The pharmaceutical,nutraceutical or veterinary composition according to claim 17,comprising the peptide of formula (I), or a pharmaceutically orveterinary acceptable salt thereof; vitamin C, vitamin B1, vitamin B2,vitamin B3, vitamin B5, vitamin B6, vitamin B8, vitamin B9, glycine,methionine, phenylalanine, proline, cysteine, glutamic acid, zincsulphate, copper sulphate, magnesium sulphate and iron sulphate togetherwith pharmaceutically, nutraceutical or veterinary acceptable excipientsand/or carriers.
 19. A kit comprising: (a) a peptide sequence of formula(I), or a pharmaceutically, nutraceutical or veterinary acceptable saltthereof; and (b) a pharmaceutical, nutraceutical or veterinarycomposition comprising vitamins, amino acids, or pharmaceutically,nutraceutical or veterinary acceptable salts thereof; pharmaceutically,nutraceutical or veterinary acceptable organic or inorganic salts ofiron, magnesium, copper and zinc-, together with pharmaceutically,nutraceutical or veterinary acceptable excipients and/or carriers.
 20. Apharmaceutical, nutraceutical or veterinary composition comprisingvitamins, amino acids, or pharmaceutically, nutraceutical or veterinaryacceptable salts thereof; pharmaceutically, nutraceutical or veterinaryacceptable organic or inorganic salts of iron, magnesium, copper andzinc-, together with pharmaceutically, nutraceutical or veterinaryacceptable excipients and/or carriers; wherein the weight ratio of ironto copper is from 25:1 to 900:1, of zinc to copper is from 30:1 to400:1, and of magnesium to copper is from 570:1 to 5800:1.
 21. Thepharmaceutical, nutraceutical or veterinary composition according toclaim 20, comprising vitamin C, vitamin B1, vitamin B2, vitamin B3,vitamin B5, vitamin B6, vitamin B8, vitamin B9, glycine, methionine,phenylalanine, proline, cysteine, glutamic acid, zinc sulphate, coppersulphate, magnesium sulphate and iron sulphate together withpharmaceutically, nutraceutical or veterinary acceptable excipientsand/or carriers.
 22. The pharmaceutical, nutraceutical or veterinarycomposition according to claim 20, comprising protein hydrolysate,thiamine, niacin, pantothenic acid, pyridoxine, biotin, folic acid,riboflavin, ascorbic acid, citric acid, myo-inositol, calcium chloride,magnesium sulphate, potassium dihydrogenphosphate, zinc sulphate, coppersulphate, and iron ethylendiaminetetraacetic chelate.